Anti-TrkB antibodies

ABSTRACT

The present invention relates to novel agonistic anti-TrkB antibodies and therapeutic and diagnostic methods and compositions for using the same.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 17, 2020, isnamed 01-3274-US-1-2020-03-17-Replacement-SL.txt and is 116,326 bytes insize.

FIELD OF THE INVENTION

This invention generally relates to agonistic anti-TrkB antibodies fordiagnostic and therapeutic use and in particular to humanized agonisticanti-TrkB antibodies. More specifically, agonistic anti-TrkB antibodiesand methods of use for the treatment of various diseases or disordersare disclosed. Pharmaceutical compositions and kits comprising theagonistic anti-TrkB antibody are also disclosed.

BACKGROUND OF THE INVENTION

Tropomyosin receptor kinase B (TrkB), also known as tyrosine receptorkinase B, or BDNF/NT-3 growth factors receptor or neurotrophic tyrosinekinase, receptor, type 2, is a protein that in humans is encoded by theNTRK2 gene (Genbank ID: 4915). TrkB is a receptor for brain-derivedneurotrophic factor (BDNF).

The neurotrophic tyrosine kinase receptor B (TrkB; gene symbol: NTRK2)is expressed by retinal neurons and glial cells. In the normal retina,TrkB signaling counteracts cell stress and promotes cell survival. Inthe diseased eye, such as in diabetic retinopathy or geographic atrophy,loss and functional impairments of retinal neurons and glial cells occurwhich cause visual impairments and vision loss. Activating TrkBsignaling above the basal level (which is reduced in diabeticretinopathy), can counteract the loss and functional impairments ofneurons and glial cells, thus improving visual function. Furthermore,TrkB activation has the potential to regenerate lost synapticconnections in the diseased eye, thereby promoting the regain of visualfunction. Upon ligand binding, TrkB undergoes homodimerization followedby autophosphorylation. Dependent on the phosphorylation sites (Y516,Y702, Y706, Y707 or Y817) different signal transduction pathways areactivated, including the activity of PLCγ1 or different subforms of AKTand ERK which regulate distinct overlapping signalling cascades inducingaxonal/neurite outgrowth, increasing synaptic plasticity, or increasingcell survival.

Agonistic anti-TrkB antibodies have been described in the US20100196390and US20100150914 as well as their proposed use in the treatment of e.g.Charcot-Marie-Tooth disease or diabetes.

However, there remains a significant need for new potent agonisticanti-TrkB antibodies that can be used to activate the TrkB pathway andthereby allow their use in therapeutic interventions of e.g.neurodegenerative and psychiatric disorders.

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies that specificallybind to human TrkB. In one aspect, the antibodies of the presentinvention have agonistic activity and induce TrkB phosphorylation and/oractivation. In another aspect, the antibodies of the present inventionare useful, for example for the treatment of eye or retinal diseasessuch as, geographic atrophy secondary to age-related maculardegeneration, diabetic retinopathy, glaucoma, and/or diabetic macularedema.

In another aspect, the present invention provides an anti-TrkB antibody,in particular a humanized anti-TrkB antibody, having one or more of theproperties described herein below.

In another aspect, an anti-TrkB antibody of the present invention bindswith high affinity to human TrkB. In an embodiment relating to thisaspect, an anti-TrkB antibody of the present invention binds to humanTrkB at a K_(D)<10 nM. In another embodiment, an anti-TrkB antibody ofthe present invention binds to human TrkB at a K_(D)<5 nM.

In another aspect, an anti-TrkB antibody of the present inventionactivates TrkB with high potency. In an embodiment relating to thisaspect, an anti-TrkB antibody of the present invention activates humanTrkB with an EC₅₀<100 pM. In a further embodiment, an anti-TrkB antibodyof the present invention activates human TrkB with an EC₅₀<50 pm.

In another aspect, an anti-TrkB antibody of the present invention ismore potent in inducing activation of TrkB downstream signaling pathwaysthan the natural TrkB ligand, BDNF. In a further aspect, an anti-TrkBantibody of the present invention regulates gene expression throughTrkB-mediated signaling pathways in a comparable pattern to that ofBDNF.

In a further aspect, an anti-TrkB antibody of the present inventionbinds a novel epitope in the extracellular domain of human TrkB. In afurther aspect, an anti-TrkB antibody of the present invention does notcross-react with TrkB from other species, in particular does notcross-react with rodent TrkB.

In one aspect, an anti-TrkB antibody of the present invention can beformulated to high concentrations for intravitreal injections into theeye.

In a further aspect, an anti-TrkB antibody of the present invention hasa low immunogenicity risk as measured by the method described in example5.

In another aspect, the CDR sequences of an anti-TrkB antibody of thepresent invention have low sequence liabilities as determined by themethod described in example 4.

In yet another aspect, an anti-TrkB antibody of the present inventiondoes not reduce BDNF induced ERK phosphorylation.

In a further aspect, an anti-TrkB antibody of the present invention isspecific for TrkB phosphorylation and/or activation and does notunspecifically phosphorylate/activate TrkA or TrkC. In another aspect,an anti-TrkB antibody does not bind unspecifically to human VEGF.

Further aspects encompass polynucleotide(s) molecule(s) encodingantibodies of the present invention, expression vectors and viralvectors as well as host cells comprising such polynucleotide(s)molecule(s), and methods of making antibodies of the present invention.The present invention further provides therapeutic uses for theantibodies of the present invention, in particular for retinal/eyediseases.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof comprising:

a light chain variable region comprising the amino acid sequence of SEQID NO: 48 (L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2);and the amino acid sequence of SEQ ID NO: 50 (L-CDR3); and

a heavy chain variable region comprising the amino acid sequence of SEQID NO: 51 (H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2);and the amino acid sequence of SEQ ID NO: 53 (H-CDR3), or

a heavy chain variable region comprising the amino acid sequence of SEQID NO: 55 (H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2);and the amino acid sequence of SEQ ID NO: 57 (H-CDR3), or

a heavy chain variable region comprising the amino acid sequence of SEQID NO: 58 (H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2);and the amino acid sequence of SEQ ID NO: 60 (H-CDR3).

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof comprising:

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 2 and SEQ ID NO: 12, respectively,

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 3 and SEQ ID NO: 13, respectively,

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 4 and SEQ ID NO: 14, respectively,

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 5 and SEQ ID NO: 15, respectively,

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 6 and SEQ ID NO: 16, respectively,

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 7 and SEQ ID NO: 17, respectively,

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 8 and SEQ ID NO: 18, respectively,

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 9 and SEQ ID NO: 19, respectively, or

a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 10 and SEQ ID NO: 20, respectively.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof comprising:

a light chain comprising the amino acid sequence of SEQ ID NO: 21 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 22 or 23,

a light chain comprising the amino acid sequence of SEQ ID NO: 24 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 25 or 26,

a light chain comprising the amino acid sequence of SEQ ID NO: 27 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 28 or 29,

a light chain comprising the amino acid sequence of SEQ ID NO: 30 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 31 or 32,

a light chain comprising the amino acid sequence of SEQ ID NO: 33 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 34 or 35,

a light chain comprising the amino acid sequence of SEQ ID NO: 36 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 37 or 38,

a light chain comprising the amino acid sequence of SEQ ID NO: 39 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 40 or 41,

a light chain comprising the amino acid sequence of SEQ ID NO: 42 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 43 or 44,or

a light chain comprising the amino acid sequence of SEQ ID NO: 45 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 46 or 47.

In a particular preferred embodiment the anti-TrkB antibody is ahumanized anti-TrkB antibody.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof for use in medicine.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof for use in the treatment ofretinal or eye diseases.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof for use in the treatment ofneural/neuronal eye or retinal diseases.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof for use in the treatment ofgeographic atrophy.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof for use in the treatment ofage-related macular degeneration or diabetic retinopathy.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising an anti-TrkB antibody or an antigen-bindingfragment thereof and a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides an anti-TrkB antibodyor an antigen-binding fragment thereof or a pharmaceutical compositioncomprising the anti-TrkB antibody or an antigen-binding fragmentthereof, wherein said antibody or antigen-binding fragment thereof isadministered by a parenteral route, intravenous route, intravitrealroute or subcutaneous route of administration.

In one embodiment, the present invention provides an isolatedpolynucleotide or polynucleotides comprising a sequence encoding a lightchain or light chain variable region of an antibody or antigen-bindingfragment thereof and a heavy chain or heavy chain variable region of anantibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides an expression vectorcomprising an isolated polynucleotide or polynucleotides encoding alight chain or light chain variable region of an antibody orantigen-binding fragment thereof and a heavy chain or heavy chainvariable region of an antibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides a viral vectorcomprising an isolated polynucleotide or polynucleotides encoding alight chain or light chain variable region of an antibody orantigen-binding fragment thereof and a heavy chain or heavy chainvariable region of an antibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides a host cell comprisingan expression vector or an isolated polynucleotide or polynucleotidesencoding a light chain or light chain variable region of an antibody orantigen-binding fragment thereof and a heavy chain or heavy chainvariable region of an antibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides a method for producingan anti-TrkB antibody or an antigen-binding fragment thereof comprising:

obtaining a host cell comprising an expression vector or an isolatedpolynucleotide or polynucleotides encoding a light chain or light chainvariable region of an antibody or antigen-binding fragment thereof and aheavy chain or heavy chain variable region of an antibody orantigen-binding fragment thereof; and cultivating the host cell.

In one embodiment, the method for producing an anti-TrkB antibody orantigen-binding fragment thereof further comprises recovering andpurifying the anti-TrkB antibody or antigen-binding fragment thereof.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least one aminoacid residue within amino acid regions 92-112, 130-143 and/or 205-219 ofthe extracellular domain of human TrkB with the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least one aminoacid residue within amino acid regions 92-112 and 130-143; or 92-112 and205-219; or 130-143 and 205-219; or 92-112 and 130-143 and 205-219 ofthe extracellular domain of human TrkB with the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least one aminoacid residue within any of the aforementioned combinations and furtheralso to at least one amino acid residue within amino acid regions313-330 and/or 348-367 of the extracellular domain of human TrkB withthe SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least one aminoacid residue within amino acid regions 92-112, 130-143, 205-219, 313-330and 348-367 of the extracellular domain of human TrkB with the SEQ IDNO: 54.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Epitope mapping of BDNF, 277-antibody, C2 or C20 antibodies tothe extracellular domain of human TrkB (SEQ ID NO: 61).

FIG. 2: qPCR expression data (y-axis) for regulated genes (x-axis) withregard to synaptic plasticity in SH-SYSY cells after treatment withBDNF, control IgG1 (anti-2,4,6-trinitrophenyl, anti-TNP) or D003antibody.

FIG. 3: Plot of the concentration of 277-antibody in ocular compartmentsas indicated and plasma after ivt injection of 1 mg of 277-antibody intorabbit eyes as a function of time.

FIG. 4: Calculated ocular PK of IgG1 antibodies. Y-axis shows thevitreal concentration and x-axis the days post ivt application. The timepost ivt injection to reach the vitreal concentration equivalent to theEC₅₀ is indicated by arrows.

FIG. 5: Study overview including animal groups and time course. At week5, a baseline ERG assessment (pre ivt application) was performed toevaluate the extent of hyperglycemia-induced neuronal dysfunction in theretina. Group 1 and Group 2 received ivt injections of an IgG1 controlantibody anti-2,4,6-trinitrophenyl (anti-TNP) which did not cause anyeffects on retinal function. Eyes of group 3 were treated with C2 (ananti-TrkB agonistic surrogate antibody). Retinal function was againevaluated at week 7 (post ivt).

FIG. 6: Changes in rod-driven b-wave implicit times relative to the meanimplicit time of the control group, at baseline (pre ivt) and 1 weekpost ivt application (see FIG. 5 for a study overview). The non-diabeticcontrol group 1 (black, n=23 eyes investigated) and the hyperglycemicgroup 2 (dotted, n=21 eyes investigated) received ivt injections with acontrol IgG1. Group 3 received ivt treatment with C2 (n=24 eyesinvestigated). Data are mean+SEM. ns, not significant; *, p<0.05; **,p<0.01; ***, p<0.001; pre ivt differences between the hyperglycemicgroups 2 and 3 and the control group 1 are significant (statisticsomitted for clarity; one-way ANOVA with Tukey's multiple comparisonstest).

FIG. 7: Light sensitivities, S, of rod-driven b-waves normalized to themean light sensitivities of the control group, S_(control), at baseline(pre ivt) or 1 week post ivt application (see FIG. 5 for a studyoverview). The non-diabetic control group 1 (black, n=23 eyesinvestigated) and the hyperglycemic group 2 (red, n=20 eyesinvestigated) received ivt injections with a control IgG1. Group 3received ivt treatment with C2 (n=24 eyes investigated). Data aremean+SEM. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; preivt differences between the hyperglycemic groups 2 and 3 and the controlgroup 1 are significant (statistics omitted for clarity; one-way ANOVAwith Tukey's multiple comparisons test).

FIG. 8: Rod-driven a-wave responses evoked by a 0.1 cd·s/m² light flash,R, normalized to the mean response of the controls, at baseline (preivt) or 1 week post ivt application (see FIG. 5 for a study overview).The non-diabetic control group 1 (black, n=23 eyes investigated) and thehyperglycemic group 2 (red, n=20 eyes investigated) received ivtinjections with a control IgG1. Group 3 received ivt treatment with C2(n=24 eyes investigated). Data are mean+SEM. ns, not significant; *,p<0.05; **, p<0.01; ***, p<0.001; pre ivt differences betweenhyperglycemic groups 2 and 3 and control group 1 are significant(statistics omitted for clarity; one-way ANOVA with Tukey's multiplecomparisons test).

FIG. 9: Changes in UV cone-driven b-wave implicit times relative to themean implicit time of the control group, at baseline (pre ivt) and 1week post ivt application (see FIG. 5 for a study overview). Thenon-diabetic control group 1 (black, n=24 eyes investigated) and thehyperglycemic group 2 (red, n=22 eyes investigated) received ivtinjections with a control IgG1. Group 3 received ivt treatment with C2(n=24 eyes investigated). Data are mean+SEM. ns, not significant; *,p<0.05; **, p<0.01; ***, p<0.001; pre ivt differences betweenhyperglycemic groups 2 and 3 and control group 1 are significant(statistics omitted for clarity; one-way ANOVA with Tukey's multiplecomparisons test).

FIG. 10: Changes in M cone-driven b-wave implicit times relative to themean implicit time of the control group, at baseline (pre ivt) and 1week post ivt application (see FIG. 5 for a study overview). Thenon-diabetic control group 1 (black, n=24 eyes investigated) and thehyperglycemic group 2 (red, n=22 eyes investigated) received ivtinjections with a control IgG1. Group 3 received ivt treatment with C2(n=24 eyes investigated). Data are mean+SEM. ns, not significant; *,p<0.05; **, p<0.01; ***, p<0.001; pre ivt differences betweenhyperglycemic groups 2 and 3 and control group 1 are significant(statistics omitted for clarity; one-way ANOVA with Tukey's multiplecomparisons test).

FIG. 11: Light sensitivities, S, of UV cone-driven b-waves normalized tothe mean light sensitivities of the control groups, S_(control), atbaseline (pre ivt) or 1 week post ivt application (see FIG. 5 for astudy overview). The non-diabetic control group 1 (black, n=22 eyesinvestigated) and the hyperglycemic group 2 (red, n=22 eyesinvestigated) received ivt injections with a control IgG1. Group 3received ivt treatment with C2 (n=21 eyes investigated). Data aremean+SEM. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; preivt differences between hyperglycemic groups 2 and 3 and control group 1are significant (statistics omitted for clarity; one-way ANOVA withTukey's multiple comparisons test).

FIG. 12: Saturating response amplitudes, R_(max), of M cone-drivenb-waves normalized to the mean saturating response amplitude ofcontrols, R_(max), control, at baseline (pre ivt) or 1 week post ivtapplication (see FIG. 5 for a study overview). The non-diabetic controlgroup 1 (black, n=24 eyes investigated) and the hyperglycemic group 2(red, n=22 eyes investigated) received ivt injections with a controlIgG1. Group 3 received ivt treatment with C2 (n=24 eyes investigated).Data are mean+SEM. ns, not significant; *, p<0.05; **, p<0.01; ***,p<0.001; pre ivt differences between hyperglycemic groups 2 and 3 andcontrol group 1 are significant (statistics omitted for clarity; one-wayANOVA with Tukey's multiple comparisons test).

FIG. 13A: ERK-phosphorylation in CHO cells expressing human TrkBreceptor after stimulation with BDNF, 277-antibody, or a combination of1 nM BDNF with increasing concentrations of 277-antibody. Data (symbols)are expressed as mean±SEM (for some points, the error bars are shorterthan the height of the symbol); connecting lines reflect non-linearregression (log(agonist) vs. response (three parameters)). Onerepresentative experiment of a series of three with independent cellbatches is shown; n.s., non-significant difference between 1 nM BDNFalone and 1 nM BDNF with the indicated 277-antibody concentrations.^(#)p<0.001 1 nM BDNF alone vs. 1 nM BDNF with indicated C2-antibodyconcentrations; one way ANOVA with multiple comparison test.

FIG. 13B: ERK-phosphorylation in CHO cells expressing human TrkBreceptor after stimulation with BDNF, C2 antibody, or a combination of 1nM BDNF with increasing concentrations of C2-antibody. Data (symbols)are expressed as mean±SEM (for some points, the error bars are shorterthan the height of the symbol); connecting lines reflect non-linearregression (log(agonist) vs. response (three parameters)). Onerepresentative experiment of a series of three with independent cellbatches is shown; n.s., non-significant difference between 1 nM BDNFalone and 1 nM BDNF with the indicated 277-antibody concentrations.^(#)p<0.001 1 nM BDNF alone vs. 1 nM BDNF with indicated C2-antibodyconcentrations; one way ANOVA with multiple comparison test.

FIG. 14: Binding of 277-antibody or C2 antibody to human or rat VEGF inan in vitro ELISA assay. Data (symbols) are expressed as mean±SEM (note:the error bars are shorter than the height of the symbol); connectinglines reflect non-linear regression (log(agonist) vs. response (threeparameters)). One representative experiment of a series of fiveindependent experiments is shown. ^(#)p<0.01 hVEGF-C2 vs. hVEGF-IsotypeIgG1 at the indicated concentrations (unpaired t-test).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The generalized structure of antibodies or immunoglobulin is well knownto those of skill in the art, these molecules are heterotetramericglycoproteins, typically of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is covalently linked to a heavy chain by one disulfide bondto form a heterodimer, and the heterotrimeric molecule is formed througha covalent disulfide linkage between the two identical heavy chains ofthe heterodimers. Although the light and heavy chains are linkedtogether by one disulfide bond, the number of disulfide linkages betweenthe two heavy chains varies by immunoglobulin isotype. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at the amino-terminus a variable domain (V_(H)=variableheavy chain), followed by three or four constant domains (C_(H1),C_(H2), C_(H3), and C_(H4)), as well as a hinge region between C_(H1)and C_(H2). Each light chain has two domains, an amino-terminal variabledomain (V_(L)=variable light chain) and a carboxy-terminal constantdomain (C_(L)). The V_(L) domain associates non-covalently with theV_(H) domain, whereas the C_(L) domain is commonly covalently linked tothe C_(H1) domain via a disulfide bond. Particular amino acid residuesare believed to form an interface between the light and heavy chainvariable domains (Chothia et al., 1985, J. Mol. Biol. 186:651-663.)

Certain domains within the variable domains differ extensively betweendifferent antibodies i.e., are “hypervariable.” These hypervariabledomains contain residues that are directly involved in the binding andspecificity of each particular antibody for its specific antigenicdeterminant. Hypervariability, both in the light chain and the heavychain variable domains, is concentrated in three segments known ascomplementarity determining regions (CDRs) or hypervariable loops(HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991,In: Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., whereasHVLs are structurally defined according to the three-dimensionalstructure of the variable domain, as described by Chothia and Lesk,1987, J. Mol. Biol. 196: 901-917. Where these two methods result inslightly different identifications of a CDR, the structural definitionis preferred. As defined by Kabat, CDR-L1 is positioned at aboutresidues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, at aboutresidues 89-97 in the light chain variable domain; CDR-H1 is positionedat about residues 31-35, CDR-H2 at about residues 50-65, and CDR-H3 atabout residues 95-102 in the heavy chain variable domain. The CDR1,CDR2, CDR3 of the heavy and light chains therefore define the unique andfunctional properties specific for a given antibody.

The three CDRs within each of the heavy and light chains are separatedby framework regions (FR), which contain sequences that tend to be lessvariable. From the amino terminus to the carboxy terminus of the heavyand light chain variable domains, the FRs and CDRs are arranged in theorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheetconfiguration of the FRs brings the CDRs within each of the chains intoclose proximity to each other as well as to the CDRs from the otherchain. The resulting conformation contributes to the antigen bindingsite (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages647-669), although not all CDR residues are necessarily directlyinvolved in antigen binding.

FR residues and Ig constant domains are not directly involved in antigenbinding, but contribute to antigen binding and/or mediate antibodyeffector function. Some FR residues are thought to have a significanteffect on antigen binding in at least three ways: by noncovalentlybinding directly to an epitope, by interacting with one or more CDRresidues, and by affecting the interface between the heavy and lightchains. The constant domains are not directly involved in antigenbinding but mediate various Ig effector functions, such as participationof the antibody in antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC) and antibody-dependent cellularphagocytosis (ADCP).

The light chains of vertebrate immunoglobulins are assigned to one oftwo clearly distinct classes, kappa (κ) and lambda (λ), based on theamino acid sequence of the constant domain. By comparison, the heavychains of mammalian immunoglobulins are assigned to one of five majorclasses, according to the sequence of the constant domains: IgA, IgD,IgE, IgG, and IgM. IgG and IgA are further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂, respectively.The heavy chain constant domains that correspond to the differentclasses of immunoglobulins are called α, δ, ε, γ, and μ, respectively.The subunit structures and three-dimensional configurations of theclasses of native immunoglobulins are well known.

The terms, “antibody”, “anti-TrkB antibody”, “humanized anti-TrkBantibody”, and “variant humanized anti-TrkB antibody” are used herein inthe broadest sense and specifically encompass monoclonal antibodies(including full length monoclonal antibodies), multispecific antibodies(e.g., bispecific antibodies), antibodies with minor modifications suchas N- or C-terminal truncations and antibody fragments such as variabledomains and other portions of antibodies that exhibit a desiredbiological activity, e.g., TrkB binding.

The term “monoclonal antibody” (mAb) refers to an antibody of apopulation of substantially homogeneous antibodies; that is, theindividual antibodies in that population are identical except fornaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic determinant, an “epitope”. Therefore, the modifier“monoclonal” is indicative of a substantially homogeneous population ofantibodies directed to the identical epitope and is not to be construedas requiring production of the antibody by any particular method. Itshould be understood that monoclonal antibodies can be made by anytechnique or methodology known in the art; including e.g., the hybridomamethod (Kohler et al., 1975, Nature 256:495), or recombinant DNA methodsknown in the art (see, e.g., U.S. Pat. No. 4,816,567), or methods ofisolation of monoclonal recombinantly produced using phage antibodylibraries, using techniques described in Clackson et al., 1991, Nature352: 624-628, and Marks et al., 1991, J. Mol. Biol. 222: 581-597.

Chimeric antibodies consist of the heavy and light chain variableregions of an antibody from one species (e.g., a non-human mammal suchas a mouse) and the heavy and light chain constant regions of anotherspecies (e.g., human) antibody and can be obtained by linking the DNAsequences encoding the variable regions of the antibody from the firstspecies (e.g., mouse) to the DNA sequences for the constant regions ofthe antibody from the second (e.g. human) species and transforming ahost with an expression vector containing the linked sequences to allowit to produce a chimeric antibody. Alternatively, the chimeric antibodyalso could be one in which one or more regions or domains of the heavyand/or light chain is identical with, homologous to, or a variant of thecorresponding sequence in a monoclonal antibody from anotherimmunoglobulin class or isotype, or from a consensus or germlinesequence. Chimeric antibodies can include fragments of such antibodies,provided that the antibody fragment exhibits the desired biologicalactivity of its parent antibody, for example binding to the same epitope(see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc.Natl. Acad. Sci. USA 81: 6851-6855).

The terms, “antibody fragment”, “antigen binding fragment”, “anti-TrkBantibody fragment”, “humanized anti-TrkB antibody fragment”, “varianthumanized anti-TrkB antibody fragment” refer to a portion of a fulllength anti-TrkB antibody, in which a variable region or a functionalcapability is retained, for example, specific TrkB epitope binding.Examples of antibody fragments include, but are not limited to, a Fab,Fab′, F(ab′)₂, Fd, Fv, scFv and scFv-Fc fragment, a diabody, a linearantibody, a single-chain antibody, a minibody, a diabody formed fromantibody fragments, and multispecific antibodies formed from antibodyfragments.

Full length antibodies can be treated with enzymes such as papain orpepsin to generate useful antibody fragments. Papain digestion is usedto produce two identical antigen-binding antibody fragments called “Fab”fragments, each with a single antigen-binding site, and a residual “Fc”fragment. The Fab fragment also contains the constant domain of thelight chain and the C_(H1) domain of the heavy chain. Pepsin treatmentyields a F(ab′)₂ fragment that has two antigen-binding sites and isstill capable of cross-linking antigen.

Fab′ fragments differ from Fab fragments by the presence of additionalresidues including one or more cysteines from the antibody hinge regionat the C-terminus of the C_(H1) domain. F(ab′)₂ antibody fragments arepairs of Fab′ fragments linked by cysteine residues in the hinge region.Other chemical couplings of antibody fragments are also known.

“Fv” fragment contains a complete antigen-recognition and binding siteconsisting of a dimer of one heavy and one light chain variable domainin tight, non-covalent association. In this configuration, the threeCDRs of each variable domain interact to define an antigen-biding siteon the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody.

A “single-chain Fv” or “scFv” antibody fragment is a single chain Fvvariant comprising the V_(H) and V_(L) domains of an antibody where thedomains are present in a single polypeptide chain. The single chain Fvis capable of recognizing and binding antigen. The scFv polypeptide mayoptionally also contain a polypeptide linker positioned between theV_(H) and V_(L) domains in order to facilitate formation of a desiredthree-dimensional structure for antigen binding by the scFv (see, e.g.,Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, Vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

Other recognized antibody fragments include those that comprise a pairof tandem Fd segments (V_(H)—C_(H1)-V_(H)-C_(H1)) to form a pair ofantigen binding regions. These “linear antibodies” can be bispecific ormonospecific as described in, for example, Zapata et al. 1995, ProteinEng. 8(10):1057-1062.

A humanized antibody or a humanized antibody fragment is a specific typeof chimeric antibody which includes an immunoglobulin amino acidsequence variant, or fragment thereof, which is capable of binding to apredetermined antigen and which, comprises one or more FRs havingsubstantially the amino acid sequence of a human immunoglobulin and oneor more CDRs having substantially the amino acid sequence of a non-humanimmunoglobulin. This non-human amino acid sequence often referred to asan “import” sequence is typically taken from an “import” antibodydomain, particularly a variable domain. In general, a humanized antibodyincludes at least the CDRs or HVLs of a non-human antibody, insertedbetween the FRs of a human heavy or light chain variable domain.

The present invention describes specific humanized anti-TrkB antibodieswhich contain CDRs derived from the murine lead D003 inserted betweenthe FRs of human germline sequence heavy and light chain variabledomains. It will be understood that certain murine FR residues may beimportant to the function of the humanized antibodies and thereforecertain of the human germline sequence heavy and light chain variabledomains residues are modified to be the same as those of thecorresponding murine sequence.

In one aspect, a humanized anti-TrkB antibody comprises substantiallyall of at least one, and typically two, variable domains (such ascontained, for example, in Fab, Fab′, F(ab′)2, Fabc, and Fv fragments)in which all, or substantially all, of the CDRs correspond to those of anon-human immunoglobulin, and specifically herein, the CDRs are murinesequences of the lead D003, and the FRs are those of a humanimmunoglobulin consensus or germline sequence. In another aspect, ahumanized anti-TrkB antibody also includes at least a portion of animmunoglobulin Fc region, typically that of a human immunoglobulin.Ordinarily, the antibody will contain both the light chain as well as atleast the variable domain of a heavy chain. The antibody also mayinclude one or more of the C_(H1), hinge, C_(H2), C_(H3), and/or C_(H4)regions of the heavy chain, as appropriate.

A humanized anti-TrkB antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG₁, IgG₂, IgG₃, IgA₄, IgA₁ and IgA₂. For example, theconstant domain can be a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theisotype is typically IgG₁. Where such cytotoxic activity is notdesirable, the constant domain may be of another isotype, e.g., IgG₂. Analternative humanized anti-TrkB antibody can comprise sequences frommore than one immunoglobulin class or isotype, and selecting particularconstant domains to optimize desired effector functions is within theordinary skill in the art. In specific embodiments, the presentinvention provides antibodies that are IgG₁ antibodies and moreparticularly, are IgG₁ antibodies in which there is a knock-out ofeffector functions.

The FRs and CDRs, or HVLs, of a humanized anti-TrkB antibody need notcorrespond precisely to the parental sequences. For example, one or moreresidues in the import CDR, or HVL, or the consensus or germline FRsequence may be altered (e.g., mutagenized) by substitution, insertionor deletion such that the resulting amino acid residue is no longeridentical to the original residue in the corresponding position ineither parental sequence but the antibody nevertheless retains thefunction of binding to TrkB. Such alteration typically will not beextensive and will be conservative alterations. Usually, at least 75% ofthe humanized antibody residues will correspond to those of the parentalconsensus or germline FR and import CDR sequences, more often at least90%, and most frequently greater than 95%, or greater than 98% orgreater than 99%.

Immunoglobulin residues that affect the interface between heavy andlight chain variable regions (“the V_(L)-V_(H) interface”) are thosethat affect the proximity or orientation of the two chains with respectto one another. Certain residues that may be involved in interchaininteractions include V_(L) residues 34, 36, 38, 44, 46, 87, 89, 91, 96,and 98 and V_(H) residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103(utilizing the numbering system set forth in Kabat et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)). U.S. Pat. No. 6,407,213 also discusses thatresidues such as V_(L) residues 43 and 85, and V_(H) residues 43 and 60also may be involved in this interaction. While these residues areindicated for human IgG only, they are applicable across species.Important antibody residues that are reasonably expected to be involvedin interchain interactions are selected for substitution into theconsensus sequence.

The terms “consensus sequence” and “consensus antibody” refer to anamino acid sequence which comprises the most frequently occurring aminoacid residue at each location in all immunoglobulins of any particularclass, isotype, or subunit structure, e.g., a human immunoglobulinvariable domain. The consensus sequence may be based on immunoglobulinsof a particular species or of many species. A “consensus” sequence,structure, or antibody is understood to encompass a consensus humansequence as described in certain embodiments, and to refer to an aminoacid sequence which comprises the most frequently occurring amino acidresidues at each location in all human immunoglobulins of any particularclass, isotype, or subunit structure. Thus, the consensus sequencecontains an amino acid sequence having at each position an amino acidthat is present in one or more known immunoglobulins, but which may notexactly duplicate the entire amino acid sequence of any singleimmunoglobulin. The variable region consensus sequence is not obtainedfrom any naturally produced antibody or immunoglobulin. Kabat et al.,1991, Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., andvariants thereof. The FRs of heavy and light chain consensus sequences,and variants thereof, provide useful sequences for the preparation ofhumanized anti-TrkB antibodies. See, for example, U.S. Pat. Nos.6,037,454 and 6,054,297.

Human germline sequences are found naturally in human population. Acombination of those germline genes generates antibody diversity.Germline antibody sequences for the light chain of the antibody comefrom conserved human germline kappa or lambda v-genes and j-genes.Similarly the heavy chain sequences come from germline v-, d- andj-genes (LeFranc, M-P, and LeFranc, G, “The Immunoglobulin Facts Book”Academic Press, 2001).

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of the antibody's natural environment are thosematerials that may interfere with diagnostic or therapeutic uses of theantibody, and can be enzymes, hormones, or other proteinaceous ornonproteinaceous solutes. In one aspect, the antibody will be purifiedto at least greater than 95% isolation by weight of antibody.

An isolated antibody includes an antibody in situ within recombinantcells in which it is produced, since at least one component of theantibody's natural environment will not be present. Ordinarily however,an isolated antibody will be prepared by at least one purification stepin which the recombinant cellular material is removed.

The term “antibody performance” refers to factors/properties thatcontribute to antibody recognition of antigen or the effectiveness of anantibody in vivo. Changes in the amino acid sequence of an antibody canaffect antibody properties such as folding, and can influence physicalfactors such as initial rate of antibody binding to antigen (k_(a)),dissociation constant of the antibody from antigen (k_(d)), affinityconstant of the antibody for the antigen (Kd), conformation of theantibody, protein stability, and half life of the antibody.

As used herein, the terms “identical” or “percent identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of nucleotides or amino acid residues that are thesame, when compared and aligned for maximum correspondence. To determinethe percent identity, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments, the two sequences that arecompared are the same length after gaps are introduced within thesequences, as appropriate (e.g., excluding additional sequence extendingbeyond the sequences being compared). For example, when variable regionsequences are compared, the leader and/or constant domain sequences arenot considered. For sequence comparisons between two sequences, a“corresponding” CDR refers to a CDR in the same location in bothsequences (e.g., CDR-H1 of each sequence).

The determination of percent identity or percent similarity between twosequences can be accomplished using a mathematical algorithm. Apreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid encoding a protein of interest. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to protein of interest. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTAdescribed in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. Alternatively, protein sequence alignment may be carried outusing the CLUSTAL W algorithm, as described by Higgins et al., 1996,Methods Enzymol. 266:383-402.

A nucleic acid sequence is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,a nucleic acid presequence or secretory leader is operably linked to anucleic acid encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide; a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in reading frame. However, enhancers are optionallycontiguous. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers can be used.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include the progenythereof. Thus, “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers.

The term “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domesticated and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, andthe like. Preferably, the mammal is human.

A “disorder”, as used herein, is any condition that would benefit fromtreatment with a humanized anti-TrkB antibody described herein. Thisincludes chronic and acute disorders or diseases including thosepathological conditions that predispose the mammal to the disorder inquestion. Non-limiting examples or disorders to be treated hereininclude eye or retinal disorders.

As used herein, the term “TrkB-associated disorder” or “TrkB-associateddisease” refers to a condition in which modification or activation ofcells expressing TrkB is indicated. A TrkB-associated disorder includesdiseases and disorders such as age-related macular degeneration,geographic atrophy, diabetic retinopathy, diabetic macular edema,retinitis pigmentosa, inherited retinal dystrophy, inherited maculardystrophy, myopic degeneration, retinal vein occlusions, retinal arteryocclusions, endophthalmitis, uveitis, cystoid macular edema, choroidalneovascular membrane secondary to any retinal diseases, opticneuropathies, glaucoma, retinal detachment, toxic retinopathy, radiationretinopathy, and traumatic retinopathy as well as prodromal andmild-to-moderate alzheimer's diseases, delaying disease progression ofpatients with Alzheimer's disease, Huntington's disease, Parkinson'sdisease, major depressive disorder, schizophrenia, cognitive impairmentassociated with schizophrenia, prevention of first-episode psychosis inindividuals with attenuated psychosis syndrome, prevention of relapse inpatients with schizophrenia, treatment-resistant depression, andmetabolic diseases like hyperphagia, obesity or metabolic syndrome.

The term “intravitreal injection” has its normal meaning in the art andrefers to introduction of an anti-TrkB antibody or antigen-bindingfragment thereof into the vitreous of a patient.

The term “subcutaneous administration” refers to introduction of ananti-TrkB antibody or antigen-binding fragment thereof under the skin ofan animal or human patient, preferable within a pocket between the skinand underlying tissue, by relatively slow, sustained delivery from adrug receptacle. Pinching or drawing the skin up and away fromunderlying tissue may create the pocket.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery is lessthan approximately 15 minutes; in another aspect, less than 5 minutes,and in still another aspect, less than 60 seconds. In yet even anotheraspect, administration is within a pocket between the skin andunderlying tissue, where the pocket may be created by pinching ordrawing the skin up and away from underlying tissue.

The term “therapeutically effective amount” is used to refer to anamount of an anti-TrkB antibody or antigen-binding fragment thereof thatrelieves or ameliorates one or more of the symptoms of the disorderbeing treated. In doing so it is that amount that has a beneficialpatient outcome. In one aspect, the therapeutically effective amount hasa neuroprotective or neuroregenerative effect. In another aspect, thetherapeutically effective amount refers to a target serum concentrationthat has been shown to be effective in, for example, slowing diseaseprogression. Efficacy can be measured in conventional ways, depending onthe condition to be treated. For example, in eye/retinal diseases ordisorders characterized by cells expressing TrkB, efficacy can bemeasured by determining the response rates, e.g. restoration of visionor by assessing the time of delay until disease progression.

The terms “treatment” and “therapy” and the like, as used herein, aremeant to include therapeutic as well as prophylactic, or suppressivemeasures for a disease or disorder leading to any clinically desirableor beneficial effect, including but not limited to alleviation or reliefof one or more symptoms, regression, slowing or cessation of progressionof the disease or disorder. Thus, for example, the term treatmentincludes the administration of an anti-TrkB antibody or antigen-bindingfragment thereof prior to or following the onset of a symptom of adisease or disorder thereby preventing or removing one or more signs ofthe disease or disorder. As another example, the term includes theadministration of an anti-TrkB antibody or antigen-binding fragmentthereof after clinical manifestation of the disease to combat thesymptoms of the disease. Further, administration of an anti-TrkBantibody or antigen-binding fragment thereof after onset and afterclinical symptoms have developed where administration affects clinicalparameters of the disease or disorder, such as the degree of tissueinjury or the amount or extent of metastasis, whether or not thetreatment leads to amelioration of the disease, comprises “treatment” or“therapy” as used herein. Moreover, as long as the compositions of theinvention either alone or in combination with another therapeutic agentalleviate or ameliorate at least one symptom of a disorder being treatedas compared to that symptom in the absence of use of the anti-TrkBantibody composition or antigen-binding fragment thereof, the resultshould be considered an effective treatment of the underlying disorderregardless of whether all the symptoms of the disorder are alleviated ornot.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

Antibodies

Described and disclosed herein are anti-TrkB antibodies, in particularhumanized anti-TrkB antibodies as well as compositions and articles ofmanufacture comprising anti-TrkB antibodies of the present invention.Also described are antigen-binding fragments of an anti-TrkB antibody.The anti-TrkB antibodies and antigen-binding fragments thereof can beused in the treatment of a variety of diseases or disorderscharacterized by reduced activity of the TrkB pathway. An anti-TrkBantibody and an antigen-binding fragment thereof each include at least aportion that specifically recognizes a TrkB epitope.

In an initial characterization the anti-TrkB murine lead D003 wasselected based on its superior antibody performance. A library ofvariants was generated by placing the CDRs of the murine lead into FRsof the human consensus heavy and light chain variable domains andfurthermore by engineering the FRs with different alterations.

This resulted in various humanized heavy and light chain variablesequences as shown below:

VL SEQUENCES D003_VL (murine lead), variable light chain, SEQ ID NO: 1DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK277-gr_VL, (humanized) variable light chain, SEQ ID NO: 2DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIK277-33_VL: (humanized) variable light chain, SEQ ID NO: 3DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIK277-35_VL: (humanized) variable light chain, SEQ ID NO: 4DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIK277-42_VL, (humanized) variable light chain, SEQ ID NO: 5DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIK277-44_VL, (humanized) variable light chain, SEQ ID NO: 6DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIK277-48_VL, (humanized) variable light chain, SEQ ID NO: 7DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIK277-51_VL, (humanized) variable light chain, SEQ ID NO: 8DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIK277-64_VL, (humanized) variable light chain, SEQ ID NO: 9DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIK277-67_VL, (humanized) variable light chain, SEQ ID NO: 10DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIK VH SEQUENCESD003_VH, (murine lead) variable heavy chain, SEQ ID NO: 11QVQLQQSGAELAKPGASVKMSCKASGYTFTGYWMHWVKQRPGQGLEWIGYINPSTDYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSRTGNYWGQGTTLTVSS277-gr_VH, (humanized) variable heavy chain, SEQ ID NO: 12QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWMGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTLVTVSS277-33_VH, (humanized) variable heavy chain, SEQ ID NO: 13QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQGTTVTVSS277-35_VH, (humanized) variable heavy chain, SEQ ID NO: 14QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSS277-42_VH, (humanized) variable heavy chain, SEQ ID NO: 15QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSS277-44_VH, (humanized) variable heavy chain, SEQ ID NO: 16QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQGTTVTVSS277-48_VH, (humanized) variable heavy chain, SEQ ID NO: 17QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSS277-51_VH, (humanized) variable heavy chain, SEQ ID NO: 18QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQRPGQGLEWIGYINPSTDYTEYNQKFKDRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSS277-64_VH, (humanized) variable heavy chain, SEQ ID NO: 19QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSS277-67_VH, (humanized) variable heavy chain, SEQ ID NO: 20QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSThe underlined sequences correspond to the CDR regions of the variablelight and heavy chain regions.

Humanized anti-TrkB antibodies of the present invention are those thathave the light and heavy chain sequences as set forth in the followingtable. IgG1-KO mutants have been made by introducing two mutations inthe Fc region, Leu232Ala and Leu233Ala to reduce effector function.

TABLE 1 Antibody Sequence SEQ ID NO: 277-gr (LightDIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNY 21 Chain, IgG1)LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-gr (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 22 Chain, IgG1)QAPGQGLEWMGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-gr (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 23 Chain, IgG1-KO)QAPGQGLEWMGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-33 (Light DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNY 24 Chain, IgG1)LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-33 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 25 Chain, IgG1)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-33 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 26 Chain, IgG1-KO)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-35 (Light DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNY 27 Chain, IgG1)LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-35 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 28 Chain, IgG1)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-35 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 29 Chain, IgG1-KO)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-42 (Light DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNY 30 Chain, IgG1)LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-42 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 31 Chain, IgG1)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-42 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 32 Chain, IgG1-KO)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-44 (Light DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNY 33 Chain, IgG1)LAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-44 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 34 Chain, IgG1)QAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-44 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 35 Chain, IgG1-KO)QAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLTSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-48 (Light DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNY 36 Chain, IgG1)LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-48 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 37 Chain, IgG1)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-48 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 38 Chain, IgG1-KO)QRPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-51 (Light DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNY 39 Chain, IgG1)LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-51 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 40 Chain, IgG1)QRPGQGLEWIGYINPSTDYTEYNQKFKDRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-51 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 41 Chain, IgG1-KO)QRPGQGLEWIGYINPSTDYTEYNQKFKDRATLTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-64 (Light DIVMTQSPDSLAVSLGERATISCKSSQSLLYSSNQKNY 42 Chain, IgG1)LAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-64 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 43 Chain, IgG1)QAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-64 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 44 Chain, IgG1-KO)QAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-67 (Light DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNY 45 Chain, IgG1)LAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC277-67 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 46 Chain, IgG1)QAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277-67 (Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVR 47 Chain, IgG1-KO)QAPGQGLEWIGYINPSTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRTGNYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG277 L-CDR1

48 277 L-CDR2

49 277 L-CDR3

50 277 H-CDR1 GYTFTGYWMH 51 (CCG) 277 H-CDR2 YINPSTDYTEYNQKFKD 52 (CCG)277 H-CDR3 SRTGNY 53 (CCG) 277 H-CDR1 GYWMH 55 (Kabat) 277 H-CDR2YINPSTDYTEYNQKFKD 56 (Kabat) 277 H-CDR3 SRTGNY 57 (Kabat) 277 H-CDR1GYTFTGY 58 (Chothia) 277 H-CDR2 NPSTDY 59 (Chothia) 277 H-CDR3 SRTGNY 60(Chothia)

Above CDRs as per the Chemical Computing Group (CCG) numbering areunderlined (Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al,Proteins 2014; 82:1599-1610). The Kabat numbering for the sequences isdenoted by the bold text and the Chothia numbering system by theitalicized text.

In one aspect, an anti-TrkB antibody of the present invention binds tohuman TrkB at a K_(D)<10 nM. In a further aspect, an anti-TrkB antibodyof the present invention binds to human TrkB at a K_(D)<5 nM. In yet afurther aspect, an anti-TrkB antibody of the present invention binds tohuman TrkB at a K_(D) of about 1 nM. Binding affinities of anti-TrkBantibodies can be determined according to the method described inexample 6.

In another aspect, an anti-TrkB antibody of the present inventioninduces TrkB phosphorylation and/or activation with high potency. In oneaspect, an anti-TrkB antibody of the present invention phosphorylateshuman TrkB with an EC₅₀<100 pM. In a further aspect, an anti-TrkBantibody of the present invention phosphorylates human TrkB with anEC₅₀<50 pM. In a further aspect, an anti-TrkB antibody of the presentinvention phosphorylates human TrkB with an EC₅₀<40 pM. In a furtheraspect, an anti-TrkB antibody of the present invention phosphorylateshuman TrkB with an EC₅₀<30 pM. In a further aspect, an anti-TrkBantibody of the present invention phosphorylates human TrkB with an EC₅₀of about 20 pM.

In another aspect, an anti-TrkB antibody of the present invention ismore potent in inducing activation of TrkB downstream signaling pathwaysthan the natural TrkB ligand BDNF. Potency of anti-TrkB antibodies canbe determined in differentiated SH-SY5Y cells according to the methoddescribed in example 8.

In a further aspect, an anti-TrkB antibody of the present inventioninduces a gene expression comparably to the natural TrkB ligand BDNF.Stimulation with agonistic anti-TrkB antibodies increases the mRNAexpression of ARC, VGF, EGR1 which are markers for increased synapticplasticity and therefore indicators that anti-TrkB antibodies act likethe natural ligand BDNF in modulating neuronal function, i.e. theincrease of neuronal survival and/or synaptic plasticity. Geneexpression patterns can be determined according to the method describedin example 10.

In yet a further aspect an anti-TrkB antibody of the present inventionprotects neurons, glial cells and/or the neurovascular unit in theretina of patients with e.g. age-related macular degeneration,geographic atrophy or diabetic retinopathy by stimulating TrkB-dependentsurvival signaling pathways and thereby providing neuroprotection.

In another aspect an anti-TrkB antibody of the present inventionregenerates axons/dendrites and/or synapses in the retina after diseaseonset in e.g. age-related macular degeneration, geographic atrophy ordiabetic retinopathy and thereby resulting in neuroregeneration.

In a further aspect, an anti-TrkB antibody of the present invention hasa low immunogenicity risk and low sequence liabilities with respect toits CDR sequences. Immunogenicity and heterogeneity are two importantaspects of a therapeutic antibody. Measurement of such aspects can bedone by the methods as described in examples 4 and 5. The antibodies ofthe present invention have been carefully engineered to have a minimumor no immunogenicity and/or heterogeneity.

In yet another aspect, an anti-TrkB antibody of the present inventiondoes not reduce BDNF induced ERK phosphorylation. For this purpose, ERKphosphorylation can be measured e.g. in CHO cells expressing human TrkBas described in Example 13. This could mean that the ERK phosphorylationinduced by endogenously expressed BDNF is not reduced when the anti-TrkBantibody of the invention is administered. It could also mean that theanti-TrkB antibody of the invention does not reduce ERK phosphorylationwhen administered prior, concurrently or subsequently after exogenouslyadministered BDNF. In some instances, an anti-TrkB antibody does notcompete with or lower BDNF induced ERK phosphorylation compared to BDNFinduction alone.

In a further aspect, an anti-TrkB antibody of the present invention isspecific for TrkB phosphorylation and/or activation and does notunspecifically phosphorylate and/or activate TrkA or TrkC.

In another aspect, an anti-TrkB antibody does not bind unspecifically tohuman VEGF and/or rat VEGF. Binding unspecifically in this context meansthat the inventive anti-TrkB antibody does not bind to human VEGF, asmeasured e.g. in an ELISA as described in Example 14. In one embodiment,the lack of unspecific binding of an anti-TrkB antibody can bedetermined by measuring a statistically significant difference (e.g.unpaired t-test, p<0.05) in binding to human VEGF between an anti-TrkBantibody and an appropriate IgG isotype control antibody. In particular,the lack of unspecific binding of the inventive anti-TrkB antibody canbe measured by determining any differences in binding to human VEGFbetween an anti-TrkB antibody and an IgG1 isotype control antibody. Aninventive anti-TrkB antibody will not differ significantly in lack ofbinding to human VEGF compared to an equally concentrated IgG1 isotypecontrol antibody up to a concentration of about 0.3 nM, 0.4 nM, 0.5 nM,0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM,50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM or 200 nM. In an alternativeembodiment the lack of unspecific binding of the inventive anti-TrkBantibody can be measured by determining the IC₅₀ binding value of theanti-TrkB antibody to either one of human or rat VEGF. In particular, aninventive anti-TrkB antibody will exhibit an IC₅₀ binding-value to humanor rat VEGF of about above 0.9 μM, preferably above 1 μM, above 2 μM,above 30, above 50, above 10 μM, above 20 μM, above 30 μM, above 40 μM,above 50 μM, above 100 μM, above 150 μM, above 200 μM, above 250 μM,above 300 μM, above 350 μM, above 400 μM, above 450 μM, above 500 μM,above 550 μM, above 600 μM, above 650 μM, above 700 μM, above 750 μM,above 800 μM, above 850 μM, or above 900 μM.

Humanization and Amino Acid Sequence Variants

Further variant anti-TrkB antibodies and antibody fragments can beengineered based on the set of CDRs identified in the murine lead D003.It is to be understood that in said variant anti-TrkB antibodies andantibody fragments the amino acid sequence of the CDRs remain unchangedbut the surrounding regions e.g. FR regions can be engineered. Aminoacid sequence variants of the anti-TrkB antibody can be prepared byintroducing appropriate nucleotide changes into the anti-TrkB antibodyDNA, or by peptide synthesis. Such variants include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the anti-TrkB antibodies of theexamples herein. Any combination of deletions, insertions, andsubstitutions is made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of thehumanized or variant anti-TrkB antibody, such as changing the number orposition of glycosylation sites.

In some embodiments, the present invention includes anti-TrkB antibodiesor antibody fragments thereof having a variable light chain and avariable heavy chain, wherein the variable light chain amino acidsequence and the variable heavy chain amino acid sequence are at least80%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequences of SEQ ID NO: 2 and 12, or 3 and 13, or 4and 14, or 5 and 15, or 6 and 16, or 7 and 17, or 8 and 18, or 9 and 19,or 10 and 20, respectively.

In some embodiments, the present invention includes anti-TrkB antibodiesor antibody fragments thereof having a light chain and a heavy chain,wherein the light chain amino acid sequence and the heavy chain aminoacid sequence are at least 80%, at least 90%, at least 95%, at least98%, or at least 99% identical to the amino acid sequences of SEQ ID NO:21 and 22 or 23; 24 and 25 or 26; 27 and 28 or 29; 30 and 31 or 32; 33and 34 or 35; 36 and 37 or 38; 39 and 40 or 41; 42 and 43 or 44; 45 and46 or 47, respectively.

In a further embodiment, the present invention includes anti-TrkBantibodies that compete for binding to TrkB with any one of thefollowing antibodies:

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 21 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 22 or 23,

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 24 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 25 or 26,

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 27 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 28 or 29,

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 30 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 31 or 32,

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 33 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 34 or 35,

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 36 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 37 or 38,

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 39 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 40 or 41,

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 42 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 43 or 44, or

An antibody having a light chain comprising the amino acid sequence ofSEQ ID NO: 45 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 46 or 47.

Another type of amino acid variant of the antibody involves altering theoriginal glycosylation pattern of the antibody. The term “altering” inthis context means deleting one or more carbohydrate moieties found inthe antibody, and/or adding one or more glycosylation sites that werenot previously present in the antibody.

In some aspects, the present invention includes nucleic acid moleculesthat encode the amino acid sequence variants of the anti-TrkB antibodiesdescribed herein. Nucleic acid molecules encoding amino acid sequencevariants of the anti-TrkB antibody are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofthe anti-TrkB antibody.

In certain embodiments, the anti-TrkB antibody is an antibody fragment.There are techniques that have been developed for the production ofantibody fragments. Fragments can be derived via proteolytic digestionof intact antibodies (see, e.g., Morimoto et al., 1992, Journal ofBiochemical and Biophysical Methods 24:107-117; and Brennan et al.,1985, Science 229:81). Alternatively, the fragments can be produceddirectly in recombinant host cells. For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (see, e.g., Carter et al., 1992, Bio/Technology10:163-167). By another approach, F(ab′)₂ fragments can be isolateddirectly from recombinant host cell culture. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

The anti-TrkB antibodies and antigen-binding fragments thereof caninclude modifications.

In certain embodiments, it may be desirable to use an anti-TrkB antibodyfragment, rather than an intact antibody. It may be desirable to modifythe antibody fragment in order to increase its serum half life. This canbe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment. In one method, the appropriateregion of the antibody fragment can be altered (e.g., mutated), or theepitope can be incorporated into a peptide tag that is then fused to theantibody fragment at either end or in the middle, for example, by DNA orpeptide synthesis. See, e.g., WO 96/32478.

In other embodiments, the present invention includes covalentmodifications of the anti-TrkB antibodies. Covalent modificationsinclude modification of cysteinyl residues, histidyl residues, lysinyland amino-terminal residues, arginyl residues, tyrosyl residues,carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginylresidues, or seryl, or threonyl residues. Another type of covalentmodification involves chemically or enzymatically coupling glycosides tothe antibody. Such modifications may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody can be introduced intothe molecule by reacting targeted amino acid residues of the antibodywith an organic derivatizing agent that is capable of reacting withselected side chains or the amino- or carboxy-terminal residues.

Removal of any carbohydrate moieties present on the antibody can beaccomplished chemically or enzymatically. Chemical deglycosylation isdescribed by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 andby Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage ofcarbohydrate moieties on antibodies can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,1987, Meth. Enzymol 138:350.

Another type of useful covalent modification comprises linking theantibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in one or more of U.S. Pat. Nos. 4,640,835, 4,496,689,4,301,144, 4,670,417, 4,791,192 and 4,179,337.

Epitope Binding

The antibodies of the invention specifically bind to native orrecombinant human TrkB. The antibodies of the present inventionrecognize specific “TrkB antigen epitope” and “TrkB epitope”. Inparticular, the antibodies of the invention bind to an epitope in theextracellular domain of human TrkB with the SEQ ID NO: 54.

The extracellular domain of human TrkB essentially comprises thefollowing sequence (SEQ ID NO.54):

CPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTGREH

As used herein, the terms “TrkB antigen epitope” and “TrkB epitope”refer to a molecule (e.g., a peptide) or a fragment of a moleculecapable of binding to an anti-TrkB antibody or antigen-binding fragmentthereof. These terms further include, for example, a TrkB antigenicdeterminant recognized by any of the antibodies or antibody fragments ofthe present invention, which has a light and heavy chain CDR combinationselected from light chain CDRs of the SEQ ID NOs 48 to 50 and heavychain CDRs of the SEQ ID NOs: 51 to 53.

TrkB antigen epitopes can be included in proteins, protein fragments,peptides or the like. The epitopes are most commonly proteins, shortoligopeptides, oligopeptide mimics (i.e., organic compounds that mimicantibody binding properties of the TrkB antigen), or combinationsthereof.

It has been found that the antibodies or antibody fragments of thepresent invention bind to unique epitopes in the extracellular domain ofhuman TrkB. Preferably, an anti-TrkB antibody or antigen-bindingfragment thereof binds to at least one amino acid residue within aminoacid regions 92-112, 130-143 and/or 205-219 of the extracellular domainof human TrkB with the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least one aminoacid residue within amino acid regions 92-112 and 130-143; or 92-112 and205-219; or 130-143 and 205-219; or 92-112 and 130-143 and 205-219 ofthe extracellular domain of human TrkB with the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least one aminoacid residue within any of the aforementioned combinations of amino acidregions and further also to at least one amino acid residue within aminoacid regions 313-330 and/or 348-367 of the extracellular domain of humanTrkB with the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least one aminoacid residue within amino acid regions 92-112, 130-143, 205-219, 313-330and 348-367 of the extracellular domain of human TrkB with the SEQ IDNO: 54.

Thus, in the context of epitope binding, the phrase “binds within aminoacid regions X-Y . . . ” means that the anti-TrkB antibody orantigen-binding fragment thereof binds to at least one amino acidresidue within the amino acid region specified in the sequence.

If for example, the anti-TrkB antibody or antigen-binding fragmentthereof binds to at least one amino acid residue within amino acidregions 92-112 or 130-143, this has the meaning that the anti-TrkBantibody or antigen-binding fragment thereof binds to at least one aminoacid residue either within amino acid regions 92-112 or 130-143.

In a further example, if the anti-TrkB antibody or antigen-bindingfragment thereof binds to at least one amino acid residue within aminoacid regions 92-112 and 130-143, this has the meaning that the anti-TrkBantibody or antigen-binding fragment thereof binds to at least one aminoacid residue within amino acid region 92-112 and also binds to at leastone amino acid residue within amino acid region 130-143 of SEQ ID NO:54.

In another aspect, an anti-TrkB antibody or antigen-binding fragmentthereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,90%, 95%, or 100% of the amino acid residues within amino acid regions92-112, 130-143 and/or 205-219 of the extracellular domain of human TrkBwith the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acidresidues within amino acid regions 92-112 and 130-143; or 92-112 and205-219; or 130-143 and 205-219; or 92-112 and 130-143 and 205-219 ofthe extracellular domain of human TrkB with the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acidresidues within any of the aforementioned combinations of amino acidregions and further also to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, or 100% of the amino acid residues within amino acidregions 313-330 and/or 348-367 of the extracellular domain of human TrkBwith the SEQ ID NO: 54.

In one embodiment, the present invention provides an anti-TrkB antibodyor antigen-binding fragment thereof that binds to at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acidresidues within amino acid regions 92-112, 130-143, 205-219, 313-330 and348-367 of the extracellular domain of human TrkB with the SEQ ID NO:54.

If for example, the anti-TrkB antibody or antigen-binding fragmentthereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,90%, 95%, or 100% amino acid residues within amino acid regions 92-112or 130-143, this has the meaning that the anti-TrkB antibody orantigen-binding fragment thereof binds to at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid residueswithin amino acid region 92-112 or binds to at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acid residueswithin amino acid region 130-143.

In a further example, if the anti-TrkB antibody or antigen-bindingfragment thereof binds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, or 100% amino acid residues within amino acidregions 92-112 and 130-143, this has the meaning that the anti-TrkBantibody or antigen-binding fragment thereof binds to at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the amino acidresidues within amino acid region 92-112 and also binds to at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% amino acidresidues within amino acid region 130-143.

Without wishing to be bound by any theory, it is believed that due tothe binding to the novel epitopes the TrkB antibody exerts its superioreffects in activating TrkB.

Polynucleotides, Vectors, Host Cells, and Recombinant Methods

Other embodiments encompass isolated polynucleotides that comprise asequence encoding an anti-TrkB antibody, vectors, and host cellscomprising the polynucleotides, and recombinant techniques forproduction of the antibody. The isolated polynucleotides can encode anydesired form of the anti-TrkB antibody including, for example, fulllength monoclonal antibodies, Fab, Fab′, F(ab′)₂, and Fv fragments,diabodies, linear antibodies, single-chain antibody molecules, andmultispecific antibodies formed from antibody fragments.

The polynucleotide(s) that comprise a sequence encoding an anti-TrkBantibody or a fragment or chain thereof can be fused to one or moreregulatory or control sequence, as known in the art, and can becontained in suitable expression vectors or host cell as known in theart. Each of the polynucleotide molecules encoding the heavy or lightchain variable domains can be independently fused to a polynucleotidesequence encoding a constant domain, such as a human constant domain,enabling the production of intact antibodies. Alternatively,polynucleotides, or portions thereof, can be fused together, providing atemplate for production of a single chain antibody.

For recombinant production, a polynucleotide encoding the antibody isinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Many suitable vectors for expressing the recombinantantibody are available. The vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The anti-TrkB antibodies can also be produced as fusion polypeptides, inwhich the antibody is fused with a heterologous polypeptide, such as asignal sequence or other polypeptide having a specific cleavage site atthe amino terminus of the mature protein or polypeptide. Theheterologous signal sequence selected is typically one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe anti-TrkB antibody signal sequence, the signal sequence can besubstituted by a prokaryotic signal sequence. The signal sequence canbe, for example, alkaline phosphatase, penicillinase, lipoprotein,heat-stable enterotoxin II leaders, and the like. For yeast secretion,the native signal sequence can be substituted, for example, with aleader sequence obtained from yeast invertase alpha-factor (includingSaccharomyces and Kluyveromyces α-factor leaders), acid phosphatase, C.albicans glucoamylase, or the signal described in WO90/13646. Inmammalian cells, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, can be used. The DNAfor such precursor region is ligated in reading frame to DNA encodingthe humanized anti-TrkB antibody.

Expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2-D. plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV, andBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a gene that encodes aselectable marker to facilitate identification of expression. Typicalselectable marker genes encode proteins that confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, or alternatively, are complement auxotrophicdeficiencies, or in other alternatives supply specific nutrients thatare not present in complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid, and hygromycin. Common selectablemarkers for mammalian cells are those that enable the identification ofcells competent to take up a nucleic acid encoding a humanized anti-TrkBantibody, such as DHFR (dihydrofolate reductase), thymidine kinase,metallothionein-I and -II (such as primate metallothionein genes),adenosine deaminase, ornithine decarboxylase, and the like. Cellstransformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity (e.g., DG44).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-TrkB antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH),can be selected by cell growth in medium containing a selection agentfor the selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199.

Where the recombinant production is performed in a yeast cell as a hostcell, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb etal., 1979, Nature 282: 39) can be used as a selectable marker. The TRP1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1(Jones, 1977, Genetics 85:12). The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626are complemented by known plasmids bearing the LEU2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis (Van den Berg, 1990, Bio/Technology8:135). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed (Fleer et al., 1991, Bio/Technology 9:968-975).

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid molecule encoding an anti-TrkB antibody or polypeptide chainthereof. Promoters suitable for use with prokaryotic hosts include phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, tryptophan (trp) promoter system, and hybrid promoters suchas the tac promoter. Other known bacterial promoters are also suitable.Promoters for use in bacterial systems also will contain a Shine-Dalgamo(S.D.) sequence operably linked to the DNA encoding the humanizedanti-TrkB antibody.

Many eukaryotic promoter sequences are known. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Inducible promoters have the additional advantage of transcriptioncontrolled by growth conditions. These include yeast promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,derivative enzymes associated with nitrogen metabolism, metallothionein,glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 73,657. Yeastenhancers also are advantageously used with yeast promoters.

Anti-TrkB antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, or from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., 1982, Nature 297:598-601, disclosingexpression of human p-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

Another useful element that can be used in a recombinant expressionvector is an enhancer sequence, which is used to increase thetranscription of a DNA encoding an anti-TrkB antibody by highereukaryotes. Many enhancer sequences are now known from mammalian genes(e.g., globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, an enhancer from a eukaryotic cell virus is used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, 1982, Nature 297:17-18 for adescription of enhancing elements for activation of eukaryoticpromoters. The enhancer may be spliced into the vector at a position 5′or 3′ to the anti-TrkB antibody-encoding sequence, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fingi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) can also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-TrkB antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein. In some embodiments, anti-TrkB antibodies can beexpressed using the CHEF system. (See, e.g., U.S. Pat. No. 5,888,809;the disclosure of which is incorporated by reference herein.)

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-TrkBantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-TrkBantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells, including, e.g.,numerous baculoviral strains and variants and corresponding permissiveinsect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori (silk worm). Avariety of viral strains for transfection are publicly available, e.g.,the L−1 variant of Autographa californica NPV and the Bm-5 strain ofBombyx mori NPV, and such viruses may be used, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

The inventive anti-TrkB antibodies or antigen-binding fragments thereofcan also be incorporated in viral vectors, i.e. the polynucleotideencoding for the anti-TrkB antibody or antigen-binding fragment thereofis introduced into the viral vector and then expressed in the body ofthe patient after infection with the virus.

In another aspect, expression of anti-TrkB is carried out in vertebratecells. The propagation of vertebrate cells in culture (tissue culture)has become routine procedure and techniques are widely available.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, (Graham etal., 1977, J. Gen Virol. 36: 59), baby hamster kidney cells (BHK, ATCCCCL 10), Chinese hamster ovary cells/-DHFR1 (CHO, Urlaub et al., 1980,Proc. Natl. Acad. Sci. USA 77: 4216; e.g., DG44), mouse sertoli cells(TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCCCRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), caninekidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCCCRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (HepG2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells(Mather et al., 1982, Annals N.Y. Acad. Sci. 383: 44-68), MRC 5 cells,FS4 cells, and human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-TrkB antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce anti-TrkB antibody described herein maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium((MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), andDulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.) aresuitable for culturing the host cells. In addition, any of the mediadescribed in one or more of Ham et al., 1979, Meth. Enz. 58: 44, Barneset al., 1980, Anal. Biochem. 102: 255, U.S. Pat. Nos. 4,767,704,4,657,866, 4,927,762, 4,560,655, 5,122,469, WO 90/103430, and WO87/00195 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as gentamicin), trace elements (defined as inorganiccompounds usually present at final concentrations in the micromolarrange), and glucose or an equivalent energy source. Other supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, the cells may bedisrupted to release protein as a first step. Particulate debris, eitherhost cells or lysed fragments, can be removed, for example, bycentrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology10:163-167 describes a procedure for isolating antibodies that aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants. A variety of methodscan be used to isolate the antibody from the host cell.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a typical purification technique. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human gamma1, gamma2, orgamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and for humangamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). A matrix towhich an affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H3) domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, reverse phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, typically performed at low salt concentrations(e.g., from about 0-0.25M salt).

Also included are nucleic acids that hybridize under low, moderate, andhigh stringency conditions, as defined herein, to all or a portion(e.g., the portion encoding the variable region) of the nucleotidesequence represented by isolated polynucleotide sequence(s) that encodean TrkB-antibody or antibody fragment. The hybridizing portion of thehybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30 or50) nucleotides in length. The hybridizing portion of the hybridizingnucleic acid is at least 80%, e.g., at least 90%, at least 95%, or atleast 98%, identical to the sequence of a portion or all of a nucleicacid encoding an anti-TrkB polypeptide (e.g., a heavy chain or lightchain variable region), or its complement. Hybridizing nucleic acids ofthe type described herein can be used, for example, as a cloning probe,a primer, e.g., a PCR primer, or a diagnostic probe.

In some embodiments, the present invention includes isolatedpolynucleotide(s) including sequences that encode an antibody orantibody fragment having a variable light chain and a variable heavychain, wherein the variable light chain amino acid sequence and thevariable heavy chain amino acid sequence are at least 80%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequences of SEQ ID NO: 2 and 12, or 3 and 13, or 4 and 14, or 5 and 15,or 6 and 16, or 7 and 17, or 8 and 18, or 9 and 19, or 10 and 20,respectively.

In some embodiments, the present invention includes isolatedpolynucleotide(s) including sequences that encode an antibody orantibody fragment having a light chain and a heavy chain, wherein thelight chain amino acid sequence and the heavy chain amino acid sequenceare at least 80%, at least 90%, at least 95%, at least 98%, or at least99% identical to the amino acid sequences of SEQ ID NO: 21 and 22 or 23;24 and 25 or 26; 27 and 28 or 29; 30 and 31 or 32; 33 and 34 or 35; 36and 37 or 38; 39 and 40 or 41; 42 and 43 or 44; 45 and 46 or 47,respectively.

It is to be understood that in said anti-TrkB antibodies and antibodyfragments the nucleic acid sequence coding for the CDRs remain unchanged(unchanged with respect to the amino acid they encode, equivalents ofthe DNA sequence due to the degeneracy of codons are possible) but thesurrounding regions e.g. FR regions can be engineered.

Non-Therapeutic Uses

The antibodies described herein are useful as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Protein A resin, using methods well known in the art. Theimmobilized antibody is contacted with a sample containing the TrkBprotein (or fragment thereof) to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the TrkB protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the TrkB protein from the antibody.

Anti-TrkB antibodies are also useful in diagnostic assays to detectand/or quantify TrkB protein, for example, detecting TrkB expression inspecific cells, tissues, or serum.

It will be advantageous in some embodiments, for example, for diagnosticpurposes to label the antibody with a detectable moiety. Numerousdetectable labels are available, including radioisotopes, fluorescentlabels, enzyme substrate labels and the like. The label may beindirectly conjugated with the antibody using various known techniques.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody can be conjugated with a small hapten (such asdigoxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).Thus, indirect conjugation of the label with the antibody can beachieved.

Exemplary radioisotopes labels include ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. Theantibody can be labeled with the radioisotope, using the techniquesdescribed in, for example, Current Protocols in Immunology, Volumes 1and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y.,Pubs. Radioactivity can be measured, for example, by scintillationcounting.

Exemplary fluorescent labels include labels derived from rare earthchelates (europium chelates) or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, andTexas Red are available. The fluorescent labels can be conjugated to theantibody via known techniques, such as those disclosed in CurrentProtocols in Immunology, supra, for example. Fluorescence can bequantified using a fluorimeter.

There are various well-characterized enzyme-substrate labels known inthe art (see, e.g., U.S. Pat. No. 4,275,149 for a review). The enzymegenerally catalyzes a chemical alteration of the chromogenic substratethat can be measured using various techniques. For example, alterationmay be a color change in a substrate that can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light that can be measured, using achemiluminometer, for example, or donates energy to a fluorescentacceptor.

Examples of enzymatic labels include luciferases such as fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such asglucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocydic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described, for example, inO'Sullivan et al., 1981, Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J.Langone & H. Van Vunakis, eds.), Academic press, N.Y., 73: 147-166.

Examples of enzyme-substrate combinations include, for example:Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor such asorthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB); alkaline phosphatase (AP) with para-Nitrophenylphosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) witha chromogenic substrate such as p-nitrophenyl-β-D-galactosidase orfluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

In another embodiment, the anti-TrkB antibody is used unlabeled anddetected with a labeled antibody that binds the anti-TrkB antibody.

The antibodies described herein may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. See, e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Diagnostic Kits

An anti-TrkB antibody can be used in a diagnostic kit, i.e., a packagedcombination of reagents in predetermined amounts with instructions forperforming the diagnostic assay. Where the antibody is labeled with anenzyme, the kit may include substrates and cofactors required by theenzyme such as a substrate precursor that provides the detectablechromophore or fluorophore. In addition, other additives may be includedsuch as stabilizers, buffers (for example a block buffer or lysisbuffer), and the like. The relative amounts of the various reagents maybe varied widely to provide for concentrations in solution of thereagents that substantially optimize the sensitivity of the assay. Thereagents may be provided as dry powders, usually lyophilized, includingexcipients that on dissolution will provide a reagent solution havingthe appropriate concentration.

Therapeutic Uses & TrkB-Associated Disorders

In another embodiment, an anti-TrkB antibody (or a functional fragmentthereof) disclosed herein is useful in the treatment of variousdisorders associated with the expression of TrkB as described herein.

The anti-TrkB antibody or antigen-binding fragment thereof isadministered by any suitable means, including intravitreal, oral,parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Inaddition, the anti-TrkB antibody is suitably administered by pulseinfusion, particularly with declining doses of the antibody. In oneaspect, the dosing is given by injections, most preferably intravenousor subcutaneous injections, depending in part on whether theadministration is brief or chronic. Preferably, the anti-TrkB antibodyis given through an intravitreal injection into the eye.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on a variety of factors such as the type of diseaseto be treated, as defined above, the severity and course of the disease,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments.

The term “suppression” is used herein in the same context as“amelioration” and “alleviation” to mean a lessening or diminishing ofone or more characteristics of the disease.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat the disorder associated withTrkB expression.

The antibody need not be, but is optionally, formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of anti-TrkBantibody present in the formulation, the type of disorder or treatment,and other factors discussed above. These are generally used in the samedosages and with administration routes as used hereinbefore or aboutfrom 1 to 99% of the heretofore employed dosages.

The agonistic anti-TrkB antibodies of the present invention stimulateTrkB signaling. Without wishing to be bound to theory it is believedthat TrkB signaling is beneficial to protect photoreceptors, otherretinal neurons including ganglion cells and retinal pigment epithelialcells from cell death in the retina. Therefore, agonistic anti-TrkBantibodies can protect retinal neurons such as photoreceptors fromdegeneration, i.e. prevent neuro-degeneration, and also increaseneuronal survival and/or plasticity, ultimately counteracting visionloss associated with either reduced TrkB activity and/or increased cellstress or apoptotic activity. Thus, the antibodies are useful fortreatment of diseases of the eye or retinal diseases, in particularneurodegenerative retinal or eye diseases. Such diseases comprise butare not limited to age-related macular degeneration, geographic atrophy,diabetic retinopathy, diabetic macular edema, retinitis pigmentosa,inherited retinal dystrophy, inherited macular dystrophy, myopicdegeneration, retinal vein occlusions, retinal artery occlusions,endophthalmitis, uveitis, cystoid macular edema, choroidal neovascularmembrane secondary to any retinal diseases, optic neuropathies,glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy,and traumatic retinopathy.

The antibodies are furthermore useful for treatment of diseases of thecentral nervous system such as prodromal and mild-to-moderatealzheimer's diseases, delaying disease progression of patients withAlzheimer's disease, Huntington's disease, Parkinson's disease, majordepressive disorder, schizophrenia, cognitive impairment associated withschizophrenia, prevention of first-episode psychosis in individuals withattenuated psychosis syndrome, prevention of relapse in patients withschizophrenia or treatment-resistant depression.

In another embodiment the antibodies may be useful for treatment ofhearing loss, in particular for cis platin induced hearing loss as wellas noise and age-related hearing loss.

In particular, the agonistic anti-TrkB antibodies of the presentinvention are indicated for treatment of non-proliferative and/orproliferative diabetic retinopathy and/or diabetic macular edema inaddition to standard of care: Dysfunction of retinal neurons andneurodegeneration are one of the major pathological events in diabeticretinopathy and diabetic macular edema (at all stages of the disease)that ultimately cause vision loss and visual dysfunction. TrkBactivation will counteract the loss and functional impairments ofretinal neurons and thereby help to maintain normal vision, to reducethe loss of visual function during the disease and potentially help toregain visual function.

Furthermore, there is the potential use of TrkB activation as an add-onto anti-VEGF treatment which will greatly enhance the therapeuticbenefits of the latter since anti-VEGF targets only vascular dysfunctionin the eye but not the neuron/glial cells system; in addition, long-termanti-VEGF treatment might cause neurodegenerative side-effects. A TrkBactivating approach as an add-on to anti-VEGF will reduce suchneurodegenerative side-effects.

The anti-TrkB antibodies or antigen-binding fragments thereof are inparticular useful for treating or preventing retinal disorders, such asdiabetic retinopathy, diabetic macular edema, geographic atrophy andglaucoma.

In a further aspect, the anti-TrkB antibodies or antigen-bindingfragments thereof are also useful for the treatment of metabolicdiseases such as hyperphagia, obesity and metabolic syndrome.

In another aspect, TrkB-antibodies of the present invention can be usedin a method for treating and/or preventing a TrkB-associated disorder,in particular geographic atrophy, comprising administering atherapeutically effective amount of an inventive TrkB-antibody to anindividual suffering from geographic atrophy, thereby ameliorating oneor more symptoms of geographic atrophy.

In yet a further aspect, agonistic TrkB-antibodies can be used in amethod for treating and/or preventing geographic atrophy, comprisingadministering a therapeutically effective amount of an agonisticTrkB-antibody to an individual suffering from geographic atrophy,thereby ameliorating one or more symptoms of geographic atrophy.

Pharmaceutical Compositions and Administration Thereof

A composition comprising an anti-TrkB antibody or an antigen-bindingfragment thereof can be administered to a subject having or at risk ofhaving an eye or retinal disease. The invention further provides for theuse of an anti-TrkB antibody or an antigen-binding fragment thereof inthe manufacture of a medicament for prevention or treatment of a TrkBdisease. The term “subject” as used herein means any mammalian patientto which an anti-TrkB antibody or an antigen-binding fragment thereofcan be administered, including, e.g., humans and non-human mammals, suchas primates, rodents, and dogs. Subjects specifically intended fortreatment using the methods described herein include humans. Theanti-TrkB antibody or an antigen-binding fragment thereof can beadministered either alone or in combination with other compositions.

Preferred antibodies for use in such pharmaceutical compositions arethose that comprise humanized antibody or antibody fragments having avariable light chain and a variable heavy chain amino acid sequence ofSEQ ID NO: 2 and 12, or 3 and 13, or 4 and 14, or 5 and 15, or 6 and 16,or 7 and 17, or 8 and 18, or 9 and 19, or 10 and 20, respectively.

Also contemplated are antibodies for use in such pharmaceuticalcompositions that comprise humanized antibody or antibody fragmentshaving a variable light chain and a variable heavy chain amino acidsequence at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO: 2 and 12,or 3 and 13, or 4 and 14, or 5 and 15, or 6 and 16, or 7 and 17, or 8and 18, or 9 and 19, or 10 and 20, respectively.

Further preferred antibodies for use in such pharmaceutical compositionsare those that comprise humanized antibody or antibody fragments havinga light chain and a heavy chain amino acid sequence of SEQ ID NO: 21 and22 or 23; 24 and 25 or 26; 27 and 28 or 29; 30 and 31 or 32; 33 and 34or 35; 36 and 37 or 38; 39 and 40 or 41; 42 and 43 or 44; 45 and 46 or47, respectively.

Further preferred antibodies for use in such pharmaceutical compositionsthat comprise humanized antibody or antibody fragments having a lightchain and a heavy chain amino acid sequence at least 80%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 21 and 22 or 23; 24 and 25 or 26; 27 and 28 or29; 30 and 31 or 32; 33 and 34 or 35; 36 and 37 or 38; 39 and 40 or 41;42 and 43 or 44; 45 and 46 or 47, respectively.

It is to be understood that in said anti-TrkB antibodies and antibodyfragments the amino acid sequence of the CDRs remain unchanged but thesurrounding regions e.g. FR regions can be engineered.

Various delivery systems are known and can be used to administer theanti-TrkB antibody or an antigen-binding fragment thereof. Methods ofintroduction include but are not limited to intravitreal, eye drops,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, and oral routes. The anti-TrkB antibody or anantigen-binding fragment thereof can be administered, for example byinfusion, bolus or injection, and can be administered together withother biologically active agents. Administration can be systemic orlocal. In preferred embodiments, the administration is by intravitrealinjection. Formulations for such injections may be prepared in, forexample, prefilled syringes.

An anti-TrkB antibody or an antigen-binding fragment thereof can beadministered as pharmaceutical compositions comprising a therapeuticallyeffective amount of the anti-TrkB antibody or an antigen-bindingfragment thereof and one or more pharmaceutically compatibleingredients.

In typical embodiments, the pharmaceutical composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous or subcutaneous administration to human beings.Typically, compositions for administration by injection are solutions insterile isotonic aqueous buffer. Where necessary, the pharmaceutical canalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where thepharmaceutical is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the pharmaceutical is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients can be mixed prior to administration.

Further, the pharmaceutical composition can be provided as apharmaceutical kit comprising (a) a container containing an anti-TrkBantibody or an antigen-binding fragment thereof in lyophilized form and(b) a second container containing a pharmaceutically acceptable diluent(e.g., sterile water) for injection. The pharmaceutically acceptablediluent can be used for reconstitution or dilution of the lyophilizedanti-TrkB antibody or antigen-binding fragment thereof. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The amount of the anti-TrkB antibody or antigen-binding fragment thereofthat is effective in the treatment or prevention of a TrkB-relateddisorder can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the stage of disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

For example, toxicity and therapeutic efficacy of the anti-TrkB antibodyor antigen-binding fragment thereof can be determined in cell culturesor experimental animals by standard pharmaceutical procedures fordetermining the ED₅₀ (the dose therapeutically effective in 50% of thepopulation). An anti-TrkB antibody or antigen-binding fragment thereofthat exhibits a large therapeutic index is preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofthe anti-TrkB antibody or antigen-binding fragment thereof typicallylies within a range of circulating concentrations that include the ED₅₀with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any anti-TrkB antibody or antigen-binding fragment thereofused in the method, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC₅₀ (i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography, ELISA and the like.

For intravitreal injection of the TrkB-antibody generally longerintervals between treatments are preferred. Due to its improved potencythe TrkB antibodies of the present invention can be administered inlonger intervals.

In one embodiment the TrkB-antibody is administered every 6 weeks,preferably every 7 weeks, also preferred every 8 weeks, furtherpreferred every 9 weeks, more preferred every 10 weeks, furtherpreferred every 11 weeks, and more preferred every 12 weeks. In afurther preferred embodiment the TrkB-antibody is administered onceevery 3 months.

In an embodiment, the TrkB-antibody is administered to subject in needthereof at an initial dose of 1-2000 mg. In another embodiment,optionally one or more subsequent doses are administered, each of whichcomprising 1-2000 mg of the TrkB-antibody.

Since the volume that can be administered to the eye is strictly limitedit is very important that an anti-TrkB antibody can be formulated tohigh concentrations. Furthermore, potency of the anti-TrkB antibody isof great importance as a potent antibody can exert its effect at evenlower doses and thereby prolong activity and also intervals betweentreatments.

Antibodies of the present invention can be formulated to very high doseswhich include, but are not limited to 20 mg/ml, 30 mg/ml, 40 mg/ml, 50mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml.

A typical dosage that can be administered to a patient is about 3mg/eye. Typical buffer components that can be used for such aformulation comprise e.g. Sodium Acetate, PS20, and Trehalose Dihydrate.

In one embodiment, the anti-TrkB antibody is formulated with 10 mMhistidine buffer, 240 mM sucrose, 0.02 w/v % polysorbate 20 at pH 5.5with a final protein concentration of 60 mg/mL.

In some embodiments, the pharmaceutical compositions comprising theanti-TrkB antibody or antigen-binding fragment thereof can furthercomprise a therapeutic agent, either conjugated or unconjugated to thebinding agent. The anti-TrkB antibody or antigen-binding fragmentthereof can be co-administered in combination with one or moretherapeutic agents for the treatment or prevention of a TrkB-relateddisease. For example, combination therapy can include anti-VEGF,anti-PDGF, or anti-ANG2.

Such combination therapy administration can have an additive orsynergistic effect on disease parameters (e.g., severity of a symptom,the number of symptoms, or frequency of relapse).

With respect to therapeutic regimens for combinatorial administration,in a specific embodiment, an anti-TrkB antibody or antigen-bindingfragment thereof is administered concurrently with a therapeutic agent.In another specific embodiment, the therapeutic agent is administeredprior or subsequent to administration of the anti-TrkB antibody orantigen-binding fragment thereof, by at least an hour and up to severalmonths, for example at least an hour, five hours, 12 hours, a day, aweek, a month, or three months, prior or subsequent to administration ofthe anti-TrkB antibody or antigen-binding fragment thereof.

Articles of Manufacture

In another aspect, an article of manufacture containing materials usefulfor the treatment of the disorders described above is included. Thearticle of manufacture comprises a container and a label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container holds a composition that is effectivefor treating the condition and may have a sterile access port. Forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle. The activeagent in the composition is the anti-TrkB antibody or theantigen-binding fragment thereof. The label on or associated with thecontainer indicates that the composition is used for treating thecondition of choice. The article of manufacture may further comprise asecond container comprising a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution, and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention.

EXAMPLES

1. Antibody Generation (Immunization)

B-cells from mice through immunization campaign were subjected toscreening using the in-house developed Single B-cell platformtechnology. Anti-TrkB binders were further narrowed down after testingfor activity, efficacy and potency in TrkB in-vitro phosphorylationassay.

2. Production of Humanized Antibodies

The murine lead D003 was selected for further optimization. The murinelead had a variable light chain corresponding to SEQ ID NO: 1 and avariable heavy chain corresponding to SEQ ID NO: 11.

To sequence optimize the TrkB murine lead D003, the most similar humangerm-line antibody sequences were selected, IGKV4-1*01, KJ2 for thelight chain and IGHV1-46*03, HJ6 for the heavy chain. A library ofvariants was generated. This resulted in the humanized light chainvariable sequences of SEQ ID NOs: 2-10 and heavy chain variablesequences of SEQ ID NOs: 12-20.

TABLE 2 277-gr_VL, (humanized) variable light chain SEQ ID NO: 2277-33_VL: (humanized) variable light chain SEQ ID NO: 3 277-35_VL:(humanized) variable light chain SEQ ID NO: 4 277-42_VL, (humanized)variable light chain SEQ ID NO: 5 277-44_VL, (humanized) variable lightchain SEQ ID NO: 6 277-48_VL, (humanized) variable light chain SEQ IDNO: 7 277-51_VL, (humanized) variable light chain SEQ ID NO: 8277-64_VL, (humanized) variable light chain SEQ ID NO: 9 277-67_VL,(humanized) variable light chain SEQ ID NO: 10 277-gr_VH, (humanized)variable heavy chain SEQ ID NO: 12 277-33_VH: (humanized) variable heavychain SEQ ID NO: 13 277-35_VH: (humanized) variable heavy chain SEQ IDNO: 14 277-42_VH, (humanized) variable heavy chain SEQ ID NO: 15277-44_VH, (humanized) variable heavy chain SEQ ID NO: 16 277-48_VH,(humanized) variable heavy chain SEQ ID NO: 17 277-51_VH, (humanized)variable heavy chain SEQ ID NO: 18 277-64_VH, (humanized) variable heavychain SEQ ID NO: 19 277-67_VH, (humanized) variable heavy chain SEQ IDNO: 20

Exemplary humanized antibodies of the present invention are those thathave the light and heavy chain sequences as set forth in the followingtable. IgG1-KO has two mutations in the Fc region, Leu234Ala andLeu235Ala to reduce effector function.

TABLE 3 277-gr (Light Chain, IgG1) 21 277-gr (Heavy Chain, IgG1) 22277-gr (Heavy Chain, IgG1-KO) 23 277-33 (Light Chain, IgG1) 24 277-33(Heavy Chain, IgG1) 25 277-33 (Heavy Chain, IgG1-KO) 26 277-35 (LightChain, IgG1) 27 277-35 (Heavy Chain, IgG1) 28 277-35 (Heavy Chain,IgG1-KO) 29 277-42 (Light Chain, IgG1) 30 277-42 (Heavy Chain, IgG1) 31277-42 (Heavy Chain, IgG1-KO) 32 277-44 (Light Chain, IgG1) 33 277-44(Heavy Chain, IgG1) 34 277-44 (Heavy Chain, IgG1-KO) 35 277-48 (LightChain, IgG1) 36 277-48 (Heavy Chain, IgG1) 37 277-48 (Heavy Chain,IgG1-KO) 38 277-51 (Light Chain, IgG1) 39 277-51 (Heavy Chain, IgG1) 40277-51 (Heavy Chain, IgG1-KO) 41 277-64 (Light Chain, IgG1) 42 277-64(Heavy Chain, IgG1) 43 277-64 (Heavy Chain, IgG1-KO) 44 277-67 (LightChain, IgG1) 45 277-67 (Heavy Chain, IgG1) 46 277-67 (Heavy Chain,IgG1-KO) 47 277 L-CDR1 48 277 L-CDR2 49 277 L-CDR3 50 277 H-CDR1 51 277H-CDR2 52 277 H-CDR3 53

3. Epitope Information

Materials

-   -   Water (Sigma Aldrich, P/N 37877-4L)    -   Acetonitrile (Sigma Aldrich, P/N 34998-4L)    -   Formic acid (Fluka, P/N 94318)    -   Urea (Sigma Aldrich, P/N 51456-500G)    -   TCEP-HCl—10 g (Thermo Scientific—Pierce, P/N 20491)    -   Sodium Phosphate Disbasic (Sigma Aldrich, P/N 57907-100G)    -   Sodium Phosphate Monobasic (Sigma Aldrich, P/N S8282-500G)    -   ACQUITY UPLC BEH C18 VanGuard Pre-column, 130 Å, 1.7 μm, 2.1        mm×5 mm (Waters Technologies Corp, 186003975)    -   Poroszyme® Immobilized Pepsin Cartridge, 2.1 mm×30 mm (Life        Technologies Corp, 2313100)    -   Acquity UPLC BEH C18 Column 1.7 um, 1 mm×50 mm (Waters,        186002344)    -   Solvent A: 0.1% Formic acid/99% water/1% acetonitrile    -   Solvent B: 0.1% Formic acid/5% water/95% acetonitrile    -   Water Buffer: H₂O 10 mM sodium phosphate pH 7.4    -   Deuterium Buffer: D₂O 10 mM sodium phosphate pH 7.4    -   Quench Buffer: Water 8 M Urea, 0.4M TCEP-HCl

In epitope mapping control samples, the antigen is run with and withoutantibody. To determine the list of antigen peptides, this protocol isfirst run using Water buffer in place of Deuterium buffer. 4 uL ofsample is mixed with 40 uL of Deuterium buffer. This mixture wasincubated at 20° C. for multiple time points (1, 2, and 4 minutes). Then40 uL of the mixture was transferred to 40 uL of 4° C. quench buffer (4MUrea, 0.4M Tcep-HCl) and mixed. 60 uL of the quenched protein isinjected, where it's digested on the pepsin column for 2 minutes byflowing 200 uL/mL of solvent A: 0.1% Formic acid/99% water/1%acetonitrile. The subsequent peptides are desalted on the VanguardPre-column for 3 minutes. The peptic peptides are sent to a BEH C18reversed phase column inside the column/valve temperature controlledcompartment. A gradient solvent system consisting of solvent A: 0.1%Formic acid/99% water/1% acetonitrile and solvent B: 0.1% Formic acid/5%water/95% acetonitrile is utilized. The percentage of solvent B isincreased from 10% to 15% at 5.1 minute, to 50% at 11 minutes, to 90% at11.5 minutes held to 12.5 minutes, to 0% B at 13 minutes held to 14minutes. The chromatographic separation took place at 4° C. at a flowrate of 180 μl/min. After chromatographic separation the sample enteredthe Thermo Scientific Orbitrap Fusion mass spectrometer operated inpositive electrospray ionization mode. The employed method includedactivation types of CID and ETD when identifying control peptides,utilizing a resolution of 120,000, a minimum signal of 10,000, anisolation width of 1.0 and a normalized collision energy of 35.0V. TheS-lens RF level was set at 60%. For control peptides data collectiontype is profile for the full MS scan and centroid for the CID MS/MSdata. For Deuterated samples, no MS/MS is collected. Data is collectedover a mass range of 280-1800 Da. For raw LC-MS/MS fragmentation dataanalysis, control samples (with CID and ETD MS/MS) were analyzed usingProteome Discover 1.4 (Thermo Scientific) and PMi Byonic (ProteinMetrics) against the given sequence to generate a list of peptides andretention times. Raw data files were preprocessed and converted to ASCIIformat using proprietary in-house SHARC software. Identified peptideswere then matched and summarized using proprietary in-house SHAFTsoftware. Epitopes were determined by differences in average mass shiftinduced by binding after Deuterium labeling. On a peptide level,protection greater than 0.4 Da was considered significant.

Results of the epitope mapping are shown for BDNF, 277-antibodies, (C2)(US20100196390), and (C20) (US20100150914) in FIG. 1. Specific bindingsites for each molecule to the extracellular domain of human TrkB withSEQ ID NO: 54 are highlighted in dark grey. Compared to the naturalligand BDNF the 277-antibodies bind to distinct new epitopes.

4. Sequence Liabilities in the CDRs

Sequences of the CDRs are checked for the presence for any potentialliabilities such as N-glycosylation sites, strong Deamidation motifs(NG, NS, NH, NA, ND, NT, NN), Aspartate isomerization motifs (DG),Fragmentation motifs (DG, DS), Cysteine. These amino acids or motifs canundergo chemical reaction and confer undesired heterogeneity to theproduct, also with the possibility of negatively impacting targetbinding and function. For these reason, it is preferred to remove suchamino acids or motifs (if any) from the CDRs.

The CDRs of the mouse lead and humanized sequences are free of suchpotential liability motifs.

TABLE 4 Antibody D003/277 C2 C20 29D7 or TAM-163 Liabilities 0 2 3 6

5. Immunogenicity

Immunogenicity of sequences is evaluated in silico with an algorithmprovided through a license by the company Epivax. EpiMatrix Treg-adjScore take into consideration T cell epitopes and Treg epitopes. Thelower the immunogenicity score, the less likely a sequence to beimmunogenic. In general, a negative score is considered low risk ofimmunogenicity, while a positive score is viewed as indication forpotential immunogenicity.

TABLE 5 VLs Epivax score VHs Epivax score 277-gr_VL −24 277-gr_VH −46277-33_VL −31 277-33_VH −32 277-35_VL −31 277-35_VH −26 277-42_VL −27277-42_VH −26 277-44_VL −14 277-44_VH −49 277-48_VL −24 277-48_VH −29277-51 _VL −31 277-51_VH −22 277-64_VL −28 277-64_VH −44 277-67_VL −14277-67_VH −44 C2_VL −2 C2_VH −21 C20_VL +9 C20_VH +20 TAM163_VL −51TAM163_VH −19

6. Affinity:

Surface Plasmon Resonance (SPR) technology was used to determine thebinding affinity of 277-antibodies, C2 (US20100196390), and C20(US20100150914), and TAM-163 (WO2015173756) to Hu-TrkB-His. Theexperiment was performed on PrateOn XPR36 instrument.

Method: The running buffer for this experiment and all dilutions (exceptwhere stated) were done in PBS-T-EDTA with 0.01% Tween20 [100 μl of 100%Tween20 was added to 2 L of PBS-T-EDTA to make final Tween 20concentration of 0.01%]. The GLM sensorchip was normalized andpre-conditioned as per the manufacturer's recommendations. Thesensorchip was activated with equal mixture of EDC/s-NHS in thehorizontal direction for 300 sec at a flow rate of 30 μl/min andimmobilized with Recombinant Protein A/G (60 μg/ml in 10 mM acetate pH4.5) in the horizontal direction for 300 sec at a flowrate of 30 μl/minresulting in ˜7100-7130 RU of Protein A/G on the surface. The sensorchipwas deactivated with 1M ethanolamine HCl in the horizontal direction for300 sec at a flowrate of 30 μl/min. The sensorchip was stabilized with18 sec of 0.85% phosphoric acid at a flowrate of 100 μl/min 3 timeshorizontally and 3 times vertically. 277-antibody (0.6 μg/ml), C2 (2.1μg/ml), C20 (1.2 μg/ml), and TAM-163 (5 μg/ml) were captured on theProtein A/G surface vertically for 70 sec at a flowrate of 30 μl/minresulting capture levels of ˜615, 1390, 780, and 3380 RU, respectively.The baseline was stabilized by injecting PBS-T-EDTA for 60 sec at aflowrate of 100 μl/min horizontally and then a second injection ofPBS-T-EDTA for 60 sec at a flowrate 100 μl/min and dissociation 120 sechorizontally. The analyte (Hu-TrkB-His) was injected horizontally overthe captured antibody for 600 sec at a flowrate of 30 μl/min and adissociation for 1800 sec. The concentrations of the analytes were 0 nM,1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, and 100 nM. The surface wasregenerated by injecting 0.85% phosphoric acid for 18 sec at a flowrateof 100 μl/min one time horizontally and one time vertically. PBS-T-EDTAwas injected for 60 sec at a flowrate of 100 μl/min one time vertically.The interspot (interactions with sensor surface) and blank (PBS-T-EDTAwith 0.01% Tween20 or 0 nM analyte) were subtracted from the raw data.Sensorgrams were then fit globally to 1:1 Langmuir binding to provideon-rate (ka), off-rate (kd), and affinity (K_(D)) values.

277-antibodies bind to Hu-TrkB-His with a ka of 1.93×10⁵ 1/Ms, a kd of3.51×10⁻⁴ 1/s, and K_(D) of 1.2 nM. C2 binds to Hu-TrkB-His with a ka of2.32×10⁻⁴ 1/Ms, a kd of 3.39×10⁻⁴1/s, and K_(D) of 10.5 nM. C20 binds toHu-TrkB-His with a ka of 1.29×10⁵ 1/Ms, a kd of 6.13×10⁻³ 1/s, and K_(D)of 47.7 nM. TAM-163 does not bind to 100 nM Hu-TrkB-His.

TABLE 6 human TrkB mAb k_(a) (1/Ms) k_(d) (1/s) K_(D) (nM)TAM-163_anti-TrkB no binding at 100 nM C20_chimeric anti-TrkB huIgG1-1.29E+05 6.13E−03 47.7 KO_EX00077781 anti-TrkB C2_HuIgG1ko 2.32E+043.39E−04 10.5 (EX00077780) Anti-TRKB 277 antibodies 1.93E+05 3.51E−041.19

7. Non-Specific Binding:

A non-specific binding assay was developed using biosensor technology todetermine if new biological entity (NBE) candidates have significantbinding to unrelated charged proteins. In this assay, the antibodies arepassed over two SPR surfaces, one coated with an unrelated negativelycharged protein and one coated with an unrelated positively chargedprotein. When an NBE candidate displays significant non-specific bindingto these surfaces, it is likely that the cause of binding is thepresence of positive or negative charged surface patches on thecandidate. Non-specific binding of NBE candidates may translate to poorpharmacokinetics (PK) and biodistribution and may also have downstreammanufacturability impacts. The results of this assay are used in thecontext of a project specific risk assessment. The purpose of the assayis to reduce the risk of poor PK, lack of tissue target distribution,and difficulty in downstream manufacturing.

Objective is to determine whether or not 277-antibodies exhibitnon-specific binding by collecting the binding sensorgrams of277-antibodies and comparing it to those of the controls to assign anon-specific binding category as either favorable (green), acceptable(yellow), or unfavorable (red).

Method:

The experiment was performed on Biacore T200. The dilution, surfacepreparation, and binding experiments were performed at 25° C. in1×HBS-EP buffer prepared from 10×HBS-EP. The flow rate for both theimmobilization protocol and binding experiment was at 5 μL/min.

To prepare the surface for the non-specific binding experiment, chickenegg white lysozyme and trypsin inhibitor type 1-S from soybean werecoupled manually to a series S CM5 chip with the surface density of3000-5000 RU using the amine coupling kit according to the manufactureinstructions. FC1 and FC2 were activated by injecting a 1:1 mixture ofEDC and NHS for 7 min. The chicken egg white lysozyme surface wasprepared on FC2 by injecting 150 μg/mL of chicken egg white lysozyme in10 mM NaOAc, pH 5.5 for 5 min. FC1 and FC2 were deactivated by injecting1 M ethanolamine-HCl for 7 min. FC1 was used as the reference surface.FC3 was activated by injecting a 1:1 mixture of EDC and HNS for 7 min.The trypsin inhibitor type 1-S from soybean surface was prepared on FC3by injecting 300 μg/mL of trypsin inhibitor type 1-S from soybean in 10mM NaOAc, pH 4.0 buffer for 20 min followed by a 7 min injection of 1 Methanolamine-HCl to deactivate the flow cell. Anti-lysozyme polyclonalantibody, anti-trypsin inhibitor antibody, and a BI negative controlwere used as controls in the assay. The controls and antibodies wereprepared at 1 μM in 1×HBS-EP buffer. The samples were injected over FC1,FC2, and FC3 surfaces with a 10 min association and 15 min dissociation.The surfaces were regenerated between each binding cycle by injecting 1min of 0.85% phosphoric acid and 30 s of 50 mM NaOH at a flow rate of 50μL/min followed by a stabilization period of 2 min with 1×HBS-EP bufferflowing over all surfaces. Anti-lysozyme polyclonal antibody andanti-trypsin inhibitor antibody were injected over the FC1, FC2, and FC3at the beginning and end of experiment. The data was collected usingBiacore T200 Control Software version 2.0.1 and analyzed using BiacoreT200 Evaluation Software version 3.0.

The binding sensorgrams of the antibodies were compared to the BInegative control sensorgrams to determine the level of non-specificbinding to chicken egg white lysozyme and trypsin inhibitor type 1-Sfrom soybean surfaces.

The data shows that 277-antibody does not significantly bind tounrelated charged proteins; specifically chicken egg white lysozyme andtrypsin inhibitor type 1-S from soybean. 277-antibody was classified asfavorable (green) when comparing the binding response and shape of thecurves in relation to the controls. Therefore, it is postulated that277-antibody will pose little risk in terms of pharmacokinetics,biodistribution, and manufacturing based on off-targeting of chargedspecies.

TABLE 7 ~4400 RU ~2300 RU Trypsin Sample Lysozyme Surface InhibitorSurface Anti-TRKB 277 antibodies No binding No binding

8. Potency (SY5Y) and Efficacy

Differentiation of SH-SY5Y Cells to a Neuronal Phenotype

SH-SY5Y (ATCC® CRL-2266™) cells are cultured in DMEM:F12 (Lonza #BE12-719F) with 15% Fetal bovine serum in a 175 cm2 tissue culture flask(Corning #353112). For cell seeding the flask is washed one time withD-PBS (Lonza #17-512Q) and the cells are detached with 37° C. pre-warmedAccutase solution (A6964 SIGMA). After 3 min incubation at roomtemperature cells can be affiliated with a 10 ml pipet and transferredto a 15 ml Falcon tube. Cells are counted and 6000 cells in 100 μlDMEM:F12 with 15% FBS per well are seeded in a collagen-I coated 96-wellplate (BD Biosciences #354407). Cells are maintained in a humidifiedincubator at 37° C., 5% CO₂. After 8 hrs, 100 μl of a 6 μM solution ofretinoic acid (Sigma # R2625) is added to each well. The final retinoicacid concentration is 3 μM. After 48 hrs and 96 hrs, an additionalretinoic acid stimulation is necessary. To this end, 100 μl medium perwell is removed and 100 μl fresh medium with 6 μM retinoic acid isadded. After 7 days of differentiation the SH-SY5Y cells have adopaminergic-like phenotype and are ready to use.

Stimulation and Lysis of Differentiated SH-SY5Y Cells

Medium from the wells was exchanged with 80 μl pre-warmed DMEM-F12 with0.2% BSA. The plate was incubated at room temperature for 15 min.Peptides and antibodies were diluted in DMEM-F12 with 0.2% BSA. Alldilutions were prepared with low-binding tips, tubes and plates. Thecells were stimulated by adding 20 μl of the correspondingpeptide/antibody for 45 min at room temperature. During the incubationtime the cell lysis buffer was prepared in a 15 ml Falcon tube on ice: 1ml 10× Triton X-100 lysis buffer 8.7 ml H₂O 1 tablet complete mini,protease inhibitor 100 μl phosphatase inhibitor cocktail 2 100 μlphosphatase inhibitor cocktail 3 100 μl 1 mM PMSF. After cellstimulation the medium was removed and 30 μl ice cold lysis buffer wasadded per well. The plate was coated with a top seal and incubated onice for 20 min. Afterwards the plate was mixed on an Eppendorf plateshaker with 500 rpm for 5 min.

For this step all materials and solutions are stored on ice. The 96-Wellplate with lysed cells is centrifuged with 235 g for 10 s and stored onice. AlphaLISA Immunoassay Buffer (PerkinElmer # AL000C) is prepared bydilution of 260 μl 10× stock solution in 2340 μl H₂O. pNTRK2 acceptorbeads (PerkinElmer # CUSM81822; beads coated by Perkin Elmer withantibody from CellSignaling CST #4621 clone C50F3) are diluted inAlphaLISA Immunoassay Buffer to a final concentration of 10 μg/ml. NTRK2biotin antibody (PerkinElmer # CUSM81820; labeled by Perkin Elmer withantibody from CellSignaling CST=#4609 clone C17F1) are diluted inAlphaLISA Immunoassay Buffer to a final concentration of 1 nM andStreptavidin-donor beads (PerkinElmer #6760002S) are diluted inAlphaLISA Immunoassay Buffer to a final concentration of 20 μg/ml. 5 μlof pNTRK2 acceptor beads and 2.5 μl of cell lysate per well istransferred to a white small volume 384-well plate (Greiner #784075)with flat bottom and centrifuged with 235 g for 10 s and stored for 45min at room temperature in the dark. After the incubation 2.5 μl NTRK2biotin antibody per well is added and the plate is centrifuged with 235g for 10 s and stored for 45 min at room temperature in the dark.Finally, 2.5 μl Streptavidin-donor beads per well is added and the plateis centrifuged with 235 g for 10 s and stored for 30 min at roomtemperature in the dark. Immediately afterwards the plate is measuredwith an EnVision plate reader using the AlphaScreen protocol (filter 570nm #244 and mirror #444).

Determination of ERK1/2 Phosphorylation

Measurement of ERK 1/2 (Thr202/Tyr204) phosphorylation were performedwith 8 μl cell lysate according manufactures protokoll (AlphaScreenSureFire p-ERK 1/2 (Thr202/Tyr204) Kit (PerkinElmer # TGRES10k))

Determination of AKT1/2/3 Phosphorylation

Measurement of Akt 1/2/3 (Thr308) phosphorylation were performed with 8μl cell lysate according manufactures protokoll ((AlphaScreen SureFirep-Akt 1/2/3 (Thr308) kit (Perkin Elmer # TGRA3S10K)).

Several TrkB antibodies of the invention were analysed for their potencyto activate TrkB as well as initiate downstream signalling. Alsoincluded were the natural ligand BDNF and the antibodies C2 and C20. TheTrkB antibodies of the present invention were on average ten times morepotent in activating TrkB than the natural ligand BDNF. Compared to theantibodies C2 and C20 the TrkB antibodies of the invention were onaverage three times to fifteen times more potent. The downstreamsignalling p-ERK and AKT1/2/3 were fully in line with these measurementsfor the TrkB activation and showed similar improved potency of theinventive TrkB antibodies.

TABLE 8 Compound EC50 pM [mean ± SD] TrkB-P BDNF 430 [±254] 277-gr(IgG1) 39 [±18] 277-gr (IgG1-KO) 46 [±27] 277-64 (IgG1) 53 [±41] 277-64(IgG1-KO) 44 [±23] C2 134 [±72] C20 654 [±180] p-ERK 1/2 (Thr202/Tyr204)BDNF 301 [±279] 277-gr (IgG1) 30 [±12] 277-gr (IgG1-KO) 47 [±42] 277-64(IgG1) 38 [±11] 277-64 (IgG1-KO) 56 [±72] C2 103 [±56] C20 238 [±36] AKT1/2/3-P (T308) BDNF 255 [±179] 277-gr (IgG1) 23 [±0] 277-gr (IgG1-KO) 39[±22] 277-64 (IgG1) 25 [±3] 277-64 (IgG1-KO) 22 [±1] C2 132 [±76] C20430 [±111]

9. Gene Expression

A next generation sequencing analysis (data not shown) was performed fordifferentiated SH-SY5Y cells after stimulation with several TrkBagonists. To validate the results the most regulated genes were chosenwith regard to synaptic plasticity and subsequently the changes wereanalyzed again via qPCR. ARC, VGF, EGR1 are known markers for synapticplasticity. Synaptic plasticity is the process by which specificpatterns of synaptic activity result in changes in synaptic strength. InFIG. 2 the stimulation of differentiated SH-SY5Y cells with BDNF oragonistic antibodies increases the amount of ARC, VGF, EGR1 mRNAexpression which can be a marker for increased synaptic plasticity. ThemRNA expression of NTRK2 is not affected by stimulation with BDNF oragonistic antibodies. The changes in expression pattern induced by BDNFand D003 are overall very similar. These results confirmed the data fromthe next generation sequencing analysis.

In specific, SH-SY5Y cells were differentiated for 7 days in 6-well cellculture plate (SIGMA # Corning CLS3516). After differentiation, cellswere stimulated in serum free DMEM:F12 with 0.2% BSA with control IgG[10 nM], BDNF [10 nM] and D003 [10 nM] for 6 hrs in a humidifiedincubator at 37° C., 5% CO2. Afterwards cells were washed once withpre-warmed 1×D-PBS and mRNA isolation was performed followedmanufactures instruction (Qiagen #74104 RNeasy Mini Kit). Thetranscription of the mRNA to cDNA was performed followed manufacturesinstruction (Qiagen #205310 QuantiTect Rev. Transcription Kit). cDNA wasused for TaqMan Real-Time PCR Assays based on 5′ Nuclease probes(ThermoFischer Scientific) on a 7900HT Fast Real-Time PCR System(Applied Biosystems). Delta CT values were calculated based on the CTvalue of ACTB (CT=18.18).

TaqMan Gene Expression assays from ThermoFischer Scientific:

-   -   ACTB—actin beta: Hs01060665_g1; Cat. #4331182    -   NTRK2—neurotrophic receptor tyrosine kinase 2: Hs00178811_m1;        Cat. #4331182    -   ARC—activity regulated cytoskeleton associated protein:        Hs01045540_g1; Cat. #4331182    -   VGF—nerve growth factor inducible: Hs00705044_s1, Cat. #4331182    -   EGR1—early growth response 1: Hs00152928_m1; Cat. #4331182

10. Rabbit Intravitreous (Ivt) Pharmacokinetic Study

New Zealand White female rabbits (1.7-2 kg body weight) wereacclimatized >15 days prior to the start of the study. Antibody 277 wasprepared sterile at 20 mg/mL and 1 mg (in 50 μL buffer: 60 mM sodiumacetate, 150 mM NaCl, pH 5.0) was injected bilaterally into the vitreousof anesthetized animals. At various time points, 2 animals wereeuthanized and ocular tissues consisting of aqueous humor, vitreous andretina were collected and frozen pending analysis. Blood samples weretaken on various time points (FIG. 3), centrifuged and plasma frozenpending analysis.

The quantification of the antibody was performed by ELISA. For eachtissue type, a calibration curve of the antibody (triplicates rangingfrom 0.5-100 ng/mL) was prepared in the same buffer as the samples andincluded in every plate. Absorption was measured at 405 nm using aSpectraMax 340PC-384 photometer and data analysis performed usingSoftMaxPro6.5.

The concentration of 277-antibody is depicted in FIG. 3 showing theconcentration in various ocular compartments as indicated and plasmaafter ivt injection of 1 mg of 277-antibody into rabbit eyes as afunction of time. The calculated ocular and plasma half-lifes are listedin the Table below.

TABLE 9 t_(1/2) (days) Vitreous Aqueous Retina Plasma 4.9 4.8 5.2 7.0

The calculated half-lives were 4.9, 4.8, 5.2, and 7.0 days in vitreous,aqueous humor, retina, and plasma, respectively. These half-lives aresimilar to those reported in the literature for the clinically usedrecombinant humanized monoclonal IgG1 antibody Avastin (anti-VEGF,bevacizumab, Bakri et al., Opthalmology, 2007), which were alsoconfirmed experimentally in-house. These results were as expected, sincethe intravitreal clearance of full length IgGs depends mainly on theirmolecular size, which is similar for our antibody 277 and Avastin.Therefore, the human PK, including the ocular half-life of antibody 277and Avastin is expected to be similar. The reported human ocularhalf-life of Avastin is 9.73±1.48 days (Hutton-Smith, 2016).

11. Ivt Injection Frequency of IgG1 Antibodies

Based on the in vivo data obtained in the pharmacokinetic study (Example10) as well as the potency data obtained in SH-SYSY cells the humanocular PK (vitreal concentration over time) of IgG1 antibodies wasinvestigated and calculated based on the well-establishedone-compartment kinetics regarding ocular clearance of biomolecules. Forthe calculation, the following parameters were used: a human ocularhalf-life of t_(1/2)=10 days as reported for Avastin and also supportedby the in vivo data shown in example 10; a molecular weight of 149 KDaas for full-length IgG1 antibodies, and an amount of ivt injectedantibody of 1 mg (see FIG. 4). Since the ocular PK is predominantlydriven by the molecular weight, the same plot of ocular PK is obtainedfor all five antibodies.

To estimate an ivt injection frequency, it was assumed that in vivoefficacy could be maintained at vitreal concentrations≥cellular EC₅₀ asdetermined from the TrkB-phosphorylation assays in SH-SY5Y cells(Example 8). The time post ivt injection to reach the vitrealconcentration equivalent to the EC₅₀ is indicated by arrows in FIG. 4.Based on this consideration, the time to reach such a vitrealconcentration after injection of 1 mg of antibody is summarized in thefollowing table for each antibody:

TABLE 10 Time post ivt to reach C_(vitreous) = EC₅₀ Compound [days]277-gr (IgG1) 154 277-gr (IgG1-KO) 151 277-64 (IgG1) 150 277-64(IgG1-KO) 152 C2 136

For example, after injecting 1 mg of C2 into the human eye, a vitrealconcentration corresponding to the EC₅₀ of C2 (TABLE 10) is reachedafter 136 days (i.e. after 136 days, the next ivt injection would beneeded). For comparison, in case of 277-64 (IgG1), this time wouldextend to 150 days post ivt injection. In this case, the next ivtinjection would be needed 14 days later as for C2. Targeting a minimalefficacious dose of values 10-fold higher than EC₅₀ would decrease thetime between ivt injections, however, the time difference in injectionfrequency between C2 and the other antibodies listed in TABLE 10 abovewould remain the same.

The injection volume that can be administered to the eye is severelylimited. At the same time repetitive injections in the eye are neededfor treatment of eye conditions. Improving the half-life of the antibodyis one way to extend the time between injection intervals but asmentioned is predominantly driven by the molecular weight of themolecule. As has been shown in vivo in example 10 and in SH-SY5Y cellsin example 8, by providing an improved and more potent antibodyinjection intervals can be extended serving the patients need to receiveless frequent injections in the eye.

12. Neuroprotective and Neuroregenerative Effects of an Agonistic TrkBAntibody in STZ-Induced Diabetic Rats

None of the 277-antibodies are rodent cross-reactive, therefore in vivoproof-of-concept and efficacy studies were performed with the TrkBactivating antibody C2 as surrogate-tool antibody to test the hypothesisthat a TrkB activating approach i) provides neuroprotection(‘neuroprotective approach’), and ii) provides reactivation ofdysfunctional neurons/circuitries (‘regeneration approach’). Bothapproaches were addressed in several studies. As animal disease modelsthe streptozotocin (STZ)-induced diabetic rat model was utilized whichis characterized by loss of pancreatic beta-cells, low insulin levels,stable hyperglycemia and consequently neuronal dysfunction in theretina.

Animal Study

Male Brown Norway rats (BN rats) were obtained from Charles River(Germany). Animals were housed in groups of 2 in IVCs at controlledtemperature and humidity conditions, with a 12 h light/dark cycle(lights out between 6 p.m. and 6 a.m.). They were fed ad libitum withpelleted diet no. 3438 from Provimi Kliba (Switzerland) and had freeaccess to tab water. Animals were acclimatized to their environment forone week before start of the study. The age of the rats was 6-8 weeks atstart of the in-life phase of the study. Hyperglycemia was induced byi.p. injections of STZ (65 mg/kg body weight; Sigma # S0130-1G). Non- orpoorly responding animals were not included into the study, i.e. animalswith blood glucose concentrations <20 mM at day 5 post STZ application.Body weight and blood glucose levels were monitored regularly. Animalswere dosed by a single ivt injection of C2 (50 μg in 4.6 μL) or thecontrol antibody anti-2,4,6-trinitrophenyl (anti-TNP) (50 μg in 4.5 μL)at day 36 post STZ application. Retinal function was assessed viaelectroretinography (ERG) recordings, and animals were sacrificed afterthe final recording by an overdose of i.p.-administered pentobarbital.

Dosing and Intravitreal Injections

The study overview is depicted in FIG. 5. For ivt injections, rats wereanesthetized with 2.5-3% isoflurane (Forene; Abbvie). A drop of 4 mg/mloxybuprocainhydrochlorid (Novesine; Omnivision) was administered as atopical local anesthesia. 4.6 μL of C2 stock solution (10.9 mg/mL;buffer: 20 mM sodium citrate, 115 mM NaCl, pH 6.0) was ivt injected intoeach eye of hyperglycemic rats (group 3, n=24 eyes, FIG. 5), resultingin an injected dose of ˜50 μg/eye. As control, 4.5 μL ofanti-anti-2,4,6-trinitrophenyl stock solution (11.2 mg/mL; buffer 20 mMsodium citrate, 115 mM NaCl, pH 6.0) was ivt injected into eyes ofnon-diabetic and diabetic control rats (group 1, n=24 eyes, and group 2,n=22 eyes, FIG. 5), resulting in an injected dose of ˜50 μg/eye. Ivtinjections were performed under a dissecting microscope. The antibodysolution was delivered with a 34-gauge needle (fitted on a 10 μlHamilton glass syringe) into the vitreous just behind the limbus, andthe general quality of injection was controlled by funduscopy (using theMicron IV Retinal Imaging Microscope, Phoenix Research Labs).

Electroretinography

Electroretinography (ERG) is a non-invasive electrophysiologicaltechnique to assess light-induced electrical activity of differentretinal neurons, and allows for quantifying different aspects of retinalfunction such as dim light or color vision. ERGs were measured as thepotential change between a corneal and a reference electrode using theEspion E3 ERG recording system (Diagnosys LLC). Prior to ERG recordings,animals were dark-adapted for at least 2 h, and anesthetized by i.p.injection of ketamine (Ketanest, ca. 100 mg/kg) and xylazin (Rompun, ca.5 mg/kg). The animals were placed on a heated stage to maintain the bodytemperature constant at 37° C. Pupils were dilated with 1%cyclopentolate-HCl and 2.5% phenylephrine. A drop of Methocel 2%solution (OmniVision) was placed on the cornea to prevent eyes fromdrying and cataract development during recordings. Recordings wereperformed simultaneously from both eyes with gold loop electrodes. Thereference electrode was a toothless alligator clip wetted with Methoceland attached to the cheek of the animal. For electrical grounding, aclip was attached to the tail of the animal. ERG signals were sampled at1 kHz and recorded with 0.15 Hz low-frequency and 500 Hz high-frequencycutoffs. The light stimuli consisted of brief full-field flashesdelivered by a set of light-emitting diodes (duration, <4 ms). Allflashes were produced by a Ganzfeld stimulator (ColorDome; Diagnosys),either in darkness or on steady green or red background light.

ERG Protocols

ERG responses were first recorded from dark-adapted animals (forisolating rod-driven responses), followed by recordings from animalsadapted to red background light (50 cd/m2, for isolating UV cone-drivenERG responses) and subsequently adapted to green background light (25.5cd/m², for isolating M cone-driven responses). In case of dark-adaptedERGs, responses were evoked by a series of flashes ranging from 1·10⁻⁵to 0.1 cd·s/m². For flashes with the luminance of 1·10⁻⁵ and 3·10⁻⁵cd·s/m², responses of 20 trials were averaged. For flashes between1·10⁻⁴ up to 0.05 cd·s/m², responses of 10 trial were averaged, and forthe final flash of 0.1 cd·s/m², 8 trials were averaged. Intervalsbetween individual flashes were chosen to ensure that the retinacompletely recovered from each flash (no indications of flash-inducedreduction of response amplitudes or shortening of implicit times). Basedon these criteria, the inter-flash intervals were 2 s for the 1·10⁻⁵ and3·10⁻⁵ cd·s/m² flashes, 5 s for flashes between 1·10⁻⁴ up to 0.05cd·s/m², and 10 s for the flash of 0.1 cd·s/m².

For recordings of UV cone-driven responses, animals were light-adaptedto the red background light for 2 min. Light responses were evoked by UVflashes of 0.02, 0.04, 0.08, 0.17, 0.35, 0.83, 1.66, 2.90, and 4.15μW/m². For recordings of M cone-driven responses, animals werelight-adapted to the green background light for 2 min. Light responseswere evoked by green flashes of 0.25, 0.1, 1.0, 5.0, 50, and 150cd·s/m². For UV and green flashes, responses of 10 trials were averagedwith inter-flash intervals of 3 s.

Data Analysis

To determine ERG b-wave amplitudes (positive depolarizing responsepredominantly originating from bipolar cells), ERG data were processedand analyzed using the MATLAB software (version R2014a; MathWorks). Theoscillatory potentials (small high-frequency wavelets superimposing theb-wave) were removed from the signals by 55 Hz fast Fourier transformlow-pass frequency filtering because oscillatory potentials can affectthe amplitude and position of the b-wave peak, especially underlight-adapted conditions. The b-wave amplitude was calculated from thebottom of the a-wave response (negative deflection mostly originatingfrom photoreceptor activity) to the peak of the b-wave peak. The b-waveimplicit time was measured as the time after the flash stimulus neededto reach the peak of the b-wave.

The amplitudes of b-wave as a function of the stimulus intensity werefitted by the following equation using a least-square fitting procedure(GraphPad Prism, Version 6.01):

$\begin{matrix}{R = \frac{R_{\max}I^{n}}{\left( {I^{n} + I_{h}^{n}} \right)}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

with R as the response amplitude, R_(max) as the saturating responseamplitude, I the flash intensity, I_(h) the semi-saturating flashintensity and n as Hill coefficient.

Light sensitivity, S, was defined as the ratio between saturatingresponse amplitude, R_(max), and semi-saturating flash intensity, I_(h).

S and R_(max) values were normalized to the mean values of non-diabeticcontrols obtained for week 5, pre ivt, and for week 7, post ivttreatment, respectively.

For analysis of rod-driven b-wave implicit times, the mean implicit timeof non-diabetic control animals was determined for b-waves evoked byflashes of 1·10⁻⁵, 3·10⁻⁵, 1·10⁻⁴ and 2·10⁻⁴ cd·s/m² (the correspondingimplicit times are very similar). For each study group, the relativechange to the mean value of controls was calculated for the flashintensities above and averaged, yielding the mean relative change inb-wave implicit time.

For analysis of UV cone-driven b-wave implicit times, the relativechanges in implicit time to the mean value of controls were calculatedfor b-wave responses evoked by flash intensities of 0.042, 0.083, 0.166and 0.348 μW/cm², and averaged.

For analysis of M cone-driven b-wave implicit times, the relativechanges in implicit time to the mean value of controls were calculatedfor b-wave responses evoked by flash intensities of 1, 5, 50 and 150cd·s/m², and averaged.

The rod-driven a-wave response was determined at the peak of the a-wave(the negative signal deflection immediately following the lightstimulus) evoked by the flash of 0.1 cd·s/m².

Results

At week 5 post diabetes induction, baseline ERG recordings wereperformed to quantify the extent of neuronal dysfunction in the retinaprior to ivt treatment (see also FIG. 5). Based on these ERG results, itwas ensured that the two diabetic (hyperglycemic) groups displayed asimilar distribution of retinal dysfunction (which received a follow-upivt treatment either with the control IgG1anti-2,4,6-trinitrophenyl—group 2—or the TrkB agonistic IgG1 C2—group3). Generally, all ivt treatments were well tolerated and no adverseevents were observed. Blood glucose was stable at ˜5 mM in non-diabeticcontrol rats (group 1, n=12) over the course of the study. InSTZ-induced diabetic animals (group 2 and 3, each n=12), blood glucoseconcentrations reached levels >20 mM after ˜4 days post STZ treatment,and did not decline below 20 mM over the course of the study. Ivttreatment with C2 or the control IgG1 anti-2,4,6-trinitrophenyl did nothave any effects on blood glucose levels.

Rod-Driven b-Wave Implicit Time:

The b-wave implicit time is a measure for the speed of the light-inducedelectrical response in the retina (predominantly on the level of bipolarcells and photoreceptor-to-bipolar cell synaptic transmission). Theimplicit time is defined as the time needed to reach the responseamplitude after light flash stimulation. The effects of hyperglycemiaand ivt treatment are summarized in FIG. 6. Changes in implicit timerelative to the mean implicit time measured from control rats werecalculated for the data obtained pre ivt treatment (baseline ERGassessment) and post ivt treatment. There was a robust increase inimplicit time by ˜14 ms in the two hyperglycemic groups pre ivttreatment, indicating a hyperglycemia-induced slow-down of b-waveresponse kinetics, i.e. a reduced speed of rod-driven retinalprocessing. Ivt injection of the control IgG1 anti-2,4,6-trinitrophenyldid not have any significant effects on implicit time in thenon-diabetic control group and the hyperglycemic group (group 1, blackbars, and group 2, dotted bars in FIG. 6, post ivt, respectively).However, ivt treatment with the TrkB activator C2 clearly resulted in adiminished delay in implicit time (group 3, grey bar in FIG. 6, postivt). On average, ivt treatment with C2 caused a significant speed-up ofb-wave responses by ˜9 ms (i.e. the delay in implicit time was shortenedfrom 14 ms down to 5 ms).

Rod-Driven b-Wave Light Sensitivity:

The light sensitivity of rod-driven b-wave ERG responses is a measurefor the number of neurons participating in the rod-driven retinalpathway, and their sensitivity towards light stimuli in order to producedepolarizing light responses. Light sensitivities were normalized to themean light sensitivities of control rats (group 1, pre and post ivttreatment) and are plotted in FIG. 7. Normalized light sensitivitieswere decreased ˜40-50% in the hyperglycemic animals (pre ivt treatment,group 2 and 3). Ivt injection of the control IgG1 did not have anysignificant effects on the light sensitivities of control rats (group 1)or hyperglycemic rats (group 2). Importantly, ivt treatment with theTrkB activator C2 significantly increased the light sensitivity by ˜30%(group 3, grey, post ivt compared to pre ivt).

Rod-Driven a-Wave Responses:

The rod-driven a-wave response is a measure for the size of rodphotoreceptor light responses evoked by a light flash. The rod-drivena-wave responses were normalized to the mean values obtained fromcontrol rats (group 1, pre and post ivt treatment) and are plotted inFIG. 8. Normalized saturating response amplitudes were decreased by ˜17%in the hyperglycemic animals (group 2 and 3, pre ivt treatment). Ivtinjection of the control IgG1 did not have any significant effects onthe normalized a-wave responses of control rats (group 1, black) orhyperglycemic rats (group 2, dotted), with the latter displaying afurther decline of the a-wave response in the course of the study.However, ivt treatment with the TrkB activator C2 (group 3, grey) couldfully prevent the further reduction of a-wave responses as observed forthe hyperglycemic group 2.

UV and M Cone-Driven b-Wave Implicit Times:

Changes in implicit time relative to the mean implicit time measuredfrom control rats were calculated for the data obtained pre ivttreatment (baseline ERG assessment) and post ivt treatment. Thisanalysis was performed for UV cone-driven b-wave implicit times (FIG. 9)and M cone-driven b-wave implicit times (FIG. 10). In case of UVcone-driven responses, there was a robust increase in implicit time by˜8 ms in the two hyperglycemic groups pre ivt treatment, whereas theincrease of M cone-driven implicit time was ˜5 ms in the hyperglycemic.These statistically significant effects indicate a hyperglycemia-inducedslow-down of b-wave response kinetics and reduced speed of cone-drivenretinal processing. Ivt injections of the control IgG1 did not have anysignificant effects on implicit time in the non-diabetic control group(group 1, black) or the hyperglycemic group (group 2, dotted), both forUV and M cone-driven b-wave implicit times. Ivt treatment with C2 (group3, grey) shortened the implicit time by ˜5 ms in case of UV cones(corresponding to a ˜50% normalization of b-wave response kinetics), andan almost complete restoration of normal b-wave response kinetics incase of M cone-driven b-waves.

UV Cone-Driven b-Wave Light Sensitivity:

The light sensitivity of UV cone-driven b-wave ERG responses wereassessed as described for rod-driven ERGs. Light sensitivities werenormalized to the mean light sensitivities of control rats (group 1, preand post ivt treatment) and are plotted in FIG. 11. Normalized lightsensitivities were decreased by ˜50% in the hyperglycemic animals atbaseline (pre ivt). Ivt injection of the control IgG1 did not have anysignificant effects on the light sensitivities, whereas ivt treatmentwith C2 significantly increased the light sensitivity to the levelobserved in healthy control rats (group 3, grey).

M Cone-Driven Saturating b-Wave Response Amplitude

The effect of hyperglycemia on the M cone-driven b-wave lightsensitivity is less pronounced than for the UV cone-driven b-waves (atthe time points investigated). Yet, a significant impairment of Mcone-driven b-wave saturating response amplitude could be observed atweek 5 after onset of hyperglycemia (˜15% reduction compared tonon-diabetic control rats, FIG. 12). Ivt treatment with C2 could restorethe saturating response amplitude to the level of controls (group 3,grey), while treatment with the control IgG1 did not have anysignificant effects (group 2, dotted).

The key findings are summarized below:

TrkB activation causes neuroprotection and preservation of retinalfunction. Neuroprotection was shown in the STZ-induced diabetic ratmodel displaying hyperglycemia-induced neuronal dysfunction. It wasshown that TrkB activation prevented the dysfunction of photoreceptors.

TrkB activation induces regain/improvement of neuronal function in theretina in the STZ-induced diabetic rat model after prolongedhyperglycemia, such as improved light response latency (i.e. shortenedERG b-wave implicit times), or improved rod- and cone-driven lightsensitivities.

In summary, the pharmacology data provide evidence for the therapeuticconcept that TrkB activation can improve/restore function of differentneuronal cell types within the eye in different disease conditions.

TrkB activation also protects photoreceptors and the outer retina asdemonstrated by the analysis of the ERG a-wave, which is a reflection ofphotoreceptor light responses. Similar to the results obtained for theERG a-wave, contrast sensitivity of the eyes of diabetic rats isdeclining in the course of persisting hyperglycemia, whereas ivttreatment with the TrkB activator C2 fully preserves contrastsensitivity. Furthermore, the improvement in ERG b-wave lightsensitivities of rod- and cone-driven light responses described above(reflecting the light sensitivities of the photoreceptor-to-bipolar cellsynaptic transmission) further strengthens the concept that TrkBactivation can improve/protect outer retina function including thephotoreceptors.

13. BDNF Function in Combination Treatments

Cultivation of CHO Cells Expressing Human TrkB Receptor

CHO cells expressing human TrkB (hTrkB) receptor (ThermoFisherScientific, # K1491) were cultured in DMEM (Lonza, # BE12-604F)supplemented with 10% fetal bovine serum, glutamax, non-essential aminoacids, 20 mM HEPES, 5 μg/ml blasticidin and 200 μg/ml zeocin. Twentyfour hours before stimulation, 5000 cells (30 μl) were seeded in eachcavity of a 384 well clear tissue culture plate (BD Falcon, #353963) andincubated in a humidified incubator at 37° C. and 5% CO₂.

Analysis of ERK Phosphorylation in CHO Cells with hTrkB Receptor

Twenty four hours after seeding, the supernatant of the CHO/hTrkB cellswas replaced with 40 μl room-temperature DMEM with 0.1% BSA but withoutother supplements. After 15 minutes, 10 μl DMEM/0.1% BSA with increasingconcentrations of BDNF (1E-13 to 3E-8 mol/l), 277-antibody (1E-13 to2.025E-8 mol/l), or C2-antibody (1E-13 to 2.025E-8 mol/l) alone or acombination of 1 nM BDNF with increasing concentrations of the 277- orC2-antibody (both 1E-13 to 2.025E-8 mol/l) was added in triplicate tostimulate ERK phosphorylation. After incubation for 45 minutes at roomtemperature, the supernatants were removed and the cells were lysed for20 minutes on wet ice in 20 μl lysis buffer per well (1× Tryton lysisbuffer (CellSignaling #9803-S), supplemented with complete mini proteaseinhibitor tablets (Roche #04693124001) and phosphatase inhibitorcocktail 2 (Sigma # P5726) and 3 (Sigma # P0044), and 1 mM PMSF (Sigma#93482)). Five microliters of the resulting lysate were used forquantification of ERK1/2 phosphorylation at T202/Y204 using acommercially available assay (Perkin Elmer # TGRES500), according to themanufacturer's instructions. Light emission of the acceptor beads,reflecting ERK phosphorylation, was recorded at 570 nm on a PerkingElmer EnVision microplate reader and prepared for presentation withGraphPad Prism (version 7), including the raw data (mean±SEM) and anon-linear regression (log(agonist) vs. response (three parameters)).As shown in FIG. 13 the 277-antibody did not reduce BDNF induced ERKphosphorylation. Rising concentrations of the C2 antibody resulted inreduction of the BDNF induced ERK phosphorylation down to the level ofC2 induced ERK phosphorylation. This may have significant impact ondosing of an anti-Trkb antibody in the eye where the antibodies areadministered in high saturating concentrations to prolong the time untilthe next administration is necessary. The 277-antibody can beadministered in high doses without interfering with existing BDNFinduced ERK phosphorylation whereas the C2 antibody reduces existingBDNF induced ERK phosphorylation down to the level of the antibody.

14. Binding to Human VEGF In Vitro ELISA Assay

For the in vitro binding assay, MaxiSorp 96 well plates (Nunc #437111)were coated over night at 4° C. with 500 ng/ml human VEGF (R&D Systems#293-VE-050) or rat VEGF (R&D Systems #564-RV-050) incarbonate/bicarbonate buffer (3.03 g/I Na₂CO₃ and 6.00 g/I NaHCO₃, pH9.6). Plates were washed three times with PBS-T (phosphate bufferedsaline (Invitrogen #70011-036) with 0.05% Tween20 (Sigma # P7949)) andthen incubated with 1% BSA (Sigma # A3059) in PBS-T for three hours atroom temperature to block unspecific binding. Plates were washed threetimes with PBS-T and then incubated with a 1:4 serial dilution of 1 μMAvastin (Roche clinic package), C2-antibody, 277-antibody, or a humanIgG1 isotype control antibody in PBS-T overnight at 4° C. Plates werewashed three times with PBS-T and then incubated with a 1:2000 dilutionof Alexa fluor 647 goat anti-human IgG (Invitrogen # A21445) for twohours at room temperature. After three additional rounds of washing withPBS-T, light emission at 647 nm (reflecting antibody binding to VEGF)was recorded on a Perking Elmer EnVision microplate reader. Data wereprepared for presentation with GraphPad Prism (version 7), including theraw data (mean±SEM) and a non-linear regression (log(agonist) vs.response (three parameters)).

As shown in FIG. 14 there was no statistically significant difference inhuman VEGF-binding between the 277 anti-TrkB antibody and the IgG1isotype control antibody at antibody concentrations of up to 0.25 μM(unpaired t-test, p>0.05). In contrast, a statistically significantdifference in human VEGF binding between C2 and the isotype controlantibody was already observed at an antibody concentration of 0.24 nM(unpaired t-test, **p<0.01).

Such an unspecific binding to VEGF may cause e.g. unwanted side-effects,make difficult the control of doses for TrkB activation, or increase theamount of antibody needed to achieve the desired effect

The invention claimed is:
 1. An anti-TrkB antibody or an antigen-bindingfragment thereof comprising: a. a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 48 (L-CDR1); the amino acidsequence of SEQ ID NO: 49 (L-CDR2); and the amino acid sequence of SEQID NO: 50 (L-CDR3); and b. a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 51 (H-CDR1); the amino acid sequenceof SEQ ID NO: 52 (H-CDR2); and the amino acid sequence of SEQ ID NO: 53(H-CDR3), or c. a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 55 (H-CDR1); the amino acid sequence of SEQ IDNO: 56 (H-CDR2); and the amino acid sequence of SEQ ID NO: 57 (H-CDR3),or d. a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 58 (H-CDR1); the amino acid sequence of SEQ ID NO: 59(H-CDR2); and the amino acid sequence of SEQ ID NO: 60 (H-CDR3).
 2. Theanti-TrkB antibody or the antigen-binding fragment thereof according toclaim 1, wherein the antibody or the antigen-binding fragment thereofcomprises: a. a variable light chain comprising an amino acid sequenceat least 80%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO: 2 and a variableheavy chain comprising an amino acid sequence at least 80%, at least90%, at least 95%, at least 98%, or at least 99% identical to the aminoacid sequence SEQ ID NO: 12, b. a variable light chain comprising anamino acid sequence at least 80%, at least 90%, at least 95%, at least98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:3 and a variable heavy chain comprising an amino acid sequence at least80%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence SEQ ID NO: 13, c. a variable light chaincomprising an amino acid sequence at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identical to the amino acid sequenceof SEQ ID NO: 4 and a variable heavy chain comprising an amino acidsequence at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence SEQ ID NO: 14, d. avariable light chain comprising an amino acid sequence at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence of SEQ ID NO: 5 and a variable heavy chaincomprising an amino acid sequence at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identical to the amino acid sequenceSEQ ID NO: 15, e. a variable light chain comprising an amino acidsequence at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO: 6 and avariable heavy chain comprising an amino acid sequence at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence SEQ ID NO: 16, f. a variable light chain comprisingan amino acid sequence at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% identical to the amino acid sequence of SEQID NO: 7 and a variable heavy chain comprising an amino acid sequence atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence SEQ ID NO: 17, g. a variable lightchain comprising an amino acid sequence at least 80%, at least 90%, atleast 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 8 and a variable heavy chain comprising an aminoacid sequence at least 80%, at least 90%, at least 95%, at least 98%, orat least 99% identical to the amino acid sequence SEQ ID NO: 18, h. avariable light chain comprising an amino acid sequence at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence of SEQ ID NO: 9 and a variable heavy chaincomprising an amino acid sequence at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identical to the amino acid sequenceSEQ ID NO: 19, or i. a variable light chain comprising an amino acidsequence at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO: 10 and avariable heavy chain comprising an amino acid sequence at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence SEQ ID NO:
 20. 3. The anti-TrkB antibody accordingto claim 1, wherein the antibody comprises: a. a light chain comprisingan amino acid sequence at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% identical to the amino acid sequence of SEQID NO: 21 and a heavy chain comprising an amino acid sequence at least80%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence of SEQ ID NO: 22 or 23, b. a light chaincomprising an amino acid sequence at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identical to the amino acid sequenceof SEQ ID NO: 24 and a heavy chain comprising an amino acid sequence atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO: 25 or 26, c. a lightchain comprising an amino acid sequence at least 80%, at least 90%, atleast 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 27 and a heavy chain comprising an amino acidsequence at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO: 28 or 29,d. a light chain comprising an amino acid sequence at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence of SEQ ID NO: 30 and a heavy chain comprising anamino acid sequence at least 80%, at least 90%, at least 95%, at least98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:31 or 32, e. a light chain comprising an amino acid sequence at least80%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence of SEQ ID NO: 33 and a heavy chain comprisingan amino acid sequence at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% identical to the amino acid sequence of SEQID NO: 34 or 35, f. a light chain comprising an amino acid sequence atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO: 36 and a heavy chaincomprising an amino acid sequence at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identical to the amino acid sequenceof SEQ ID NO: 37 or 38, g. a light chain comprising an amino acidsequence at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO: 39 and aheavy chain comprising an amino acid sequence at least 80%, at least90%, at least 95%, at least 98%, or at least 99% identical to the aminoacid sequence of SEQ ID NO: 40 or 41, h. a light chain comprising anamino acid sequence at least 80%, at least 90%, at least 95%, at least98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:42 and a heavy chain comprising an amino acid sequence at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence of SEQ ID NO: 43 or 44, i. a light chain comprisingan amino acid sequence at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% identical to the amino acid sequence of SEQID NO: 45 and a heavy chain comprising an amino acid sequence at least80%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence of SEQ ID NO: 46 or
 47. 4. The anti-TrkBantibody or the antigen-binding fragment thereof according to claim 1,wherein the antibody or the antigen-binding fragment thereof comprises:a. a variable light chain and a variable heavy chain comprising theamino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 12, respectively, b.a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 3 and SEQ ID NO: 13, respectively, c. avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 4 and SEQ ID NO: 14, respectively, d. avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 5 and SEQ ID NO: 15, respectively, e. avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 6 and SEQ ID NO: 16, respectively, f. avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 7 and SEQ ID NO: 17, respectively, g. avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 8 and SEQ ID NO: 18, respectively, h. avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 9 and SEQ ID NO: 19, respectively, or i. avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 10 and SEQ ID NO: 20, respectively.
 5. Theanti-TrkB antibody according to claim 1, wherein the antibody comprises:a. a light chain comprising the amino acid sequence of SEQ ID NO: 21 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 22 or 23,b. a light chain comprising the amino acid sequence of SEQ ID NO: 24 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 25 or 26,c. a light chain comprising the amino acid sequence of SEQ ID NO: 27 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 28 or 29,d. a light chain comprising the amino acid sequence of SEQ ID NO: 30 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 31 or 32,e. a light chain comprising the amino acid sequence of SEQ ID NO: 33 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 34 or 35,f. a light chain comprising the amino acid sequence of SEQ ID NO: 36 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 37 or 38,g. a light chain comprising the amino acid sequence of SEQ ID NO: 39 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 40 or 41,h. a light chain comprising the amino acid sequence of SEQ ID NO: 42 anda heavy chain comprising the amino acid sequence of SEQ ID NO: 43 or 44,or i. a light chain comprising the amino acid sequence of SEQ ID NO: 45and a heavy chain comprising the amino acid sequence of SEQ ID NO: 46 or47, respectively.
 6. The anti-TrkB antibody according to claim 1,wherein the antibody comprises: a light chain comprising the amino acidsequence of SEQ ID NO: 21 and a heavy chain comprising the amino acidsequence of SEQ ID NO: 22 or
 23. 7. The anti-TrkB antibody according toclaim 1, wherein the antibody comprises: a light chain comprising theamino acid sequence of SEQ ID NO: 24 and a heavy chain comprising theamino acid sequence of SEQ ID NO: 25 or
 26. 8. The anti-TrkB antibodyaccording to claim 1, wherein the antibody comprises: a light chaincomprising the amino acid sequence of SEQ ID NO: 27 and a heavy chaincomprising the amino acid sequence of SEQ ID NO: 28 or
 29. 9. Theanti-TrkB antibody according to claim 1, wherein the antibody comprises:a light chain comprising the amino acid sequence of SEQ ID NO: 30 and aheavy chain comprising the amino acid sequence of SEQ ID NO: 31 or 32.10. The anti-TrkB antibody according to claim 1, wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 33 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 34 or
 35. 11. The anti-TrkB antibody according to claim 1, whereinthe antibody comprises: a light chain comprising the amino acid sequenceof SEQ ID NO: 36 and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 37 or
 38. 12. The anti-TrkB antibody according to claim 1,wherein the antibody comprises: a light chain comprising the amino acidsequence of SEQ ID NO: 39 and a heavy chain comprising the amino acidsequence of SEQ ID NO: 40 or
 41. 13. The anti-TrkB antibody according toclaim 1, wherein the antibody comprises: a light chain comprising theamino acid sequence of SEQ ID NO: 42 and a heavy chain comprising theamino acid sequence of SEQ ID NO: 43 or
 44. 14. The anti-TrkB antibodyaccording to claim 1, wherein the antibody comprises: a light chaincomprising the amino acid sequence of SEQ ID NO: 45 and a heavy chaincomprising the amino acid sequence of SEQ ID NO: 46 or
 47. 15. Apharmaceutical composition comprising an antibody or an antigen-bindingfragment thereof according to claim 1 and a pharmaceutically acceptablecarrier.
 16. An isolated polynucleotide or polynucleotides comprising asequence encoding a light chain or light chain variable region of anantibody or antigen-binding fragment thereof and a heavy chain or heavychain variable region of an antibody or antigen-binding fragment thereofaccording to claim
 1. 17. An expression vector comprising the isolatedpolynucleotide or polynucleotides of claim
 16. 18. A viral vectorcomprising the isolated polynucleotide or polynucleotides of claim 16.19. A cultured host cell comprising an isolated polynucleotide orpolynucleotides according to claim
 16. 20. A method for producing ananti-TrkB antibody or antigen-binding fragment thereof comprising: a.obtaining a host cell according to claim 19; and b. cultivating the hostcell.
 21. An anti-TrkB antibody or an antigen-binding fragment thereofcomprising: a. a light chain variable region comprising the amino acidsequence of SEQ ID NO: 48 (L-CDR1); the amino acid sequence of SEQ IDNO: 49 (L-CDR2); and the amino acid sequence of SEQ ID NO: 50 (L-CDR3);and b. a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 51 (H-CDR1); the amino acid sequence of SEQ ID NO: 52(H-CDR2); and the amino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 55 (H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); andthe amino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibody orthe antigen-binding fragment thereof comprises: a variable light chainand a variable heavy chain comprising the amino acid sequences of SEQ IDNO: 2 and SEQ ID NO:
 12. 22. An anti-TrkB antibody or an antigen-bindingfragment thereof comprising: a. a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 48 (L-CDR1); the amino acidsequence of SEQ ID NO: 49 (L-CDR2); and the amino acid sequence of SEQID NO: 50 (L-CDR3); and b. a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 51 (H-CDR1); the amino acid sequenceof SEQ ID NO: 52 (H-CDR2); and the amino acid sequence of SEQ ID NO: 53(H-CDR3), or c. a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 55 (H-CDR1); the amino acid sequence of SEQ IDNO: 56 (H-CDR2); and the amino acid sequence of SEQ ID NO: 57 (H-CDR3),or d. a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 58 (H-CDR1); the amino acid sequence of SEQ ID NO:59(H-CDR2); and the amino acid sequence of SEQ ID NO: 60 (H-CDR3);wherein the antibody or the antigen-binding fragment thereof comprises:a variable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 3 and SEQ ID NO:
 13. 23. An anti-TrkBantibody or an antigen-binding fragment thereof comprising: a. a lightchain variable region comprising the amino acid sequence of SEQ ID NO:48 (L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibody orthe antigen-binding fragment thereof comprises: a variable light chainand a variable heavy chain comprising the amino acid sequences of SEQ IDNO: 4 and SEQ ID NO:
 14. 24. An anti-TrkB antibody or an antigen-bindingfragment thereof comprising: a. a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 48 (L-CDR1); the amino acidsequence of SEQ ID NO: 49 (L-CDR2); and the amino acid sequence of SEQID NO: 50 (L-CDR3); and b. a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 51 (H-CDR1); the amino acid sequenceof SEQ ID NO: 52 (H-CDR2); and the amino acid sequence of SEQ ID NO: 53(H-CDR3), or c. a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 55 (H-CDR1); the amino acid sequence of SEQ IDNO: 56 (H-CDR2); and the amino acid sequence of SEQ ID NO: 57 (H-CDR3),or d. a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 58 (H-CDR1); the amino acid sequence of SEQ ID NO: 59(H-CDR2); and the amino acid sequence of SEQ ID NO: 60 (H-CDR3); whereinthe antibody or the antigen-binding fragment thereof comprises: avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 5 and SEQ ID NO:
 15. 25. An anti-TrkBantibody or an antigen-binding fragment thereof comprising: a. a lightchain variable region comprising the amino acid sequence of SEQ ID NO:48 (L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibody orthe antigen-binding fragment thereof comprises: a variable light chainand a variable heavy chain comprising the amino acid sequences of SEQ IDNO: 6 and SEQ ID NO:
 16. 26. An anti-TrkB antibody or an antigen-bindingfragment thereof comprising: a. a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 48 (L-CDR1); the amino acidsequence of SEQ ID NO: 49 (L-CDR2); and the amino acid sequence of SEQID NO: 50 (L-CDR3); and b. a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 51 (H-CDR1); the amino acid sequenceof SEQ ID NO: 52 (H-CDR2); and the amino acid sequence of SEQ ID NO: 53(H-CDR3), or c. a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 55 (H-CDR1); the amino acid sequence of SEQ IDNO: 56 (H-CDR2); and the amino acid sequence of SEQ ID NO: 57 (H-CDR3),or d. a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 58 (H-CDR1); the amino acid sequence of SEQ ID NO: 59(H-CDR2); and the amino acid sequence of SEQ ID NO: 60 (H-CDR3); whereinthe antibody or the antigen-binding fragment thereof comprises: avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 7 and SEQ ID NO:
 17. 27. An anti-TrkBantibody or an antigen-binding fragment thereof comprising: a. a lightchain variable region comprising the amino acid sequence of SEQ ID NO:48 (L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibody orthe antigen-binding fragment thereof comprises: a variable light chainand a variable heavy chain comprising the amino acid sequences of SEQ IDNO: 8 and SEQ ID NO:
 18. 28. An anti-TrkB antibody or an antigen-bindingfragment thereof comprising: a. a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 48 (L-CDR1); the amino acidsequence of SEQ ID NO: 49 (L-CDR2); and the amino acid sequence of SEQID NO: 50 (L-CDR3); and b. a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 51 (H-CDR1); the amino acid sequenceof SEQ ID NO: 52 (H-CDR2); and the amino acid sequence of SEQ ID NO: 53(H-CDR3), or c. a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 55 (H-CDR1); the amino acid sequence of SEQ IDNO: 56 (H-CDR2); and the amino acid sequence of SEQ ID NO: 57 (H-CDR3),or d. a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 58 (H-CDR1); the amino acid sequence of SEQ ID NO: 59(H-CDR2); and the amino acid sequence of SEQ ID NO: 60 (H-CDR3); whereinthe antibody or the antigen-binding fragment thereof comprises: avariable light chain and a variable heavy chain comprising the aminoacid sequences of SEQ ID NO: 9 and SEQ ID NO:
 19. 29. An anti-TrkBantibody or an antigen-binding fragment thereof comprising: a. a lightchain variable region comprising the amino acid sequence of SEQ ID NO:48 (L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibody orthe antigen-binding fragment thereof comprises: a variable light chainand a variable heavy chain comprising the amino acid sequences of SEQ IDNO: 10 and SEQ ID NO:
 20. 30. An anti-TrkB antibody comprising: a. alight chain variable region comprising the amino acid sequence of SEQ IDNO: 48 (L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); andthe amino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 21 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 22 or
 23. 31. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 24 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 25 or
 26. 32. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 27 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 28 or
 29. 33. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 30 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 31 or
 32. 34. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 33 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 34 or
 35. 35. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 36 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 37 or
 38. 36. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 39 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 40 or
 41. 37. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 42 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 43 or
 44. 38. An anti-TrkB antibody comprising: a. a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(L-CDR1); the amino acid sequence of SEQ ID NO: 49 (L-CDR2); and theamino acid sequence of SEQ ID NO: 50 (L-CDR3); and b. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 52 (H-CDR2); and theamino acid sequence of SEQ ID NO: 53 (H-CDR3), or c. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 55(H-CDR1); the amino acid sequence of SEQ ID NO: 56 (H-CDR2); and theamino acid sequence of SEQ ID NO: 57 (H-CDR3), or d. a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 58(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); and theamino acid sequence of SEQ ID NO: 60 (H-CDR3); wherein the antibodycomprises: a light chain comprising the amino acid sequence of SEQ IDNO: 45 and a heavy chain comprising the amino acid sequence of SEQ IDNO: 46 or 47.